Compositions and methods

ABSTRACT

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/054,864 filed Aug. 3, 2018 (allowed), which is a continuation of U.S. application Ser. No. 15/847,623 filed Dec. 19, 2017, now U.S. Pat. No. 10,064,901, issued Sep. 4, 2018, which is a continuation of U.S. application Ser. No. 15/415,745 filed Jan. 25, 2017, now U.S. Pat. No. 9,855,303, issued Jan. 2, 2018, which is a continuation of U.S. application Ser. No. 14/884,655 filed Oct. 15, 2015, now U.S. Pat. No. 9,585,921, issued Mar. 7, 2017, which is a continuation of U.S. application Ser. No. 14/313,828 filed Jun. 24, 2014, now U.S. Pat. No. 9,180,147, issued Nov. 10, 2015, which is a divisional of U.S. application Ser. No. 14/197,044, filed Mar. 4, 2014, now U.S. Pat. No. 9,011,834, issued Apr. 21, 2015, which is a continuation of International Application No. PCT/US2014/014745, filed Feb. 4, 2014, which claims priority to U.S. Provisional Application No. 61/760,584, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,585, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,574, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,606, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/926,918, filed Jan. 13, 2014. These applications are all incorporated by reference in their entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing with 2043 sequences submitted electronically as a text file named 4268-029000D_SL_ST25.txt, created on Apr. 5, 2021, with a size of 4,324 kilobytes. The sequence listing is incorporated by reference.

BACKGROUND

Mammals are colonized by microbes in the gastrointestinal (GI) tract, on the skin, and in other epithelial and tissue niches such as the oral cavity, eye surface and vagina. The gastrointestinal tract harbors an abundant and diverse microbial community. It is a complex system, providing an environment or niche for a community of many different species or organisms, including diverse strains of bacteria. Hundreds of different species may form a commensal community in the GI tract in a healthy person, and this complement of organisms evolves from the time of birth to ultimately form a functionally mature microbial population by about 3 years of age. Interactions between microbial strains in these populations and between microbes and the host, e.g. the host immune system, shape the community structure, with availability of and competition for resources affecting the distribution of microbes. Such resources may be food, location and the availability of space to grow or a physical structure to which the microbe may attach. For example, host diet is involved in shaping the GI tract flora.

A healthy microbiota provides the host with multiple benefits, including colonization resistance to a broad spectrum of pathogens, essential nutrient biosynthesis and absorption, and immune stimulation that maintains a healthy gut epithelium and an appropriately controlled systemic immunity. In settings of ‘dysbiosis’ or disrupted symbiosis, microbiota functions can be lost or deranged, resulting in increased susceptibility to pathogens, altered metabolic profiles, or induction of proinflammatory signals that can result in local or systemic inflammation or autoimmunity. Thus, the intestinal microbiota plays a significant role in the pathogenesis of many diseases and disorders, including a variety of pathogenic infections of the gut. For instance, subjects become more susceptible to pathogenic infections when the normal intestinal microbiota has been disturbed due to use of broad-spectrum antibiotics. Many of these diseases and disorders are chronic conditions that significantly decrease a subject's quality of life and can be ultimately fatal.

Manufacturers of probiotics have asserted that their preparations of bacteria promote mammalian health by preserving the natural microflora in the GI tract and reinforcing the normal controls on aberrant immune responses. See, e.g., U.S. Pat. No. 8,034,601. Probiotics, however, have been limited to a very narrow group of genera and a correspondingly limited number of species; as such, they do not adequately replace the missing natural microflora of the GI tract in many situations.

Thus practitioners have a need for a method of populating a subject's gastrointestinal tract with a diverse and useful selection of microbiota in order to alter a dysbiosis.

Therefore, in response to the need for durable, efficient, and effective compositions and methods for treatment of GI diseases by way of restoring or enhancing microbiota functions, we address these and other shortcomings of the prior art by providing compositions and methods for treating subjects.

SUMMARY OF THE INVENTION

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of 16S rRNA gene and denotes the coordinates of hypervariable regions 1-9 (V1-V9). Coordinates of V1-V9 are 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294, and 1435-1465 respectively, based on numbering using E. coli system of nomenclature defined by Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene (16S rRNA) from Escherichia coli, PNAS 75(10):4801-4805 (1978). FIG. 1B highlights in bold the nucleotide sequences for each hypervariable region in the exemplary reference E. coli 16S sequence described by Brosius et al. FIG. 1B discloses SEQ ID NO: 2043.

FIG. 2 shows a photograph of a CsCl gradient demonstrating the spore separation from other residual habitat material.

FIG. 3 shows three phase contrast image demonstrating the progressive enrichment of spores from a fecal suspension; ethanol treated, CsCl purified spore preparation; and an ethanol treated, CsCl purified, sucrose purified spore preparation.

FIG. 4 shows a set of survival curves demonstrating efficacy of the spore population in a mouse prophylaxis model of C. difficile.

FIG. 5 provides a set of survival curves demonstrating efficacy of the spore population in a hamster relapse prevention model of C. difficile.

FIG. 6 demonstrates the cell viability under a variety of ethanol and heat treatments for varying lengths of time.

FIG. 7 demonstrates cell survivability from four donor fecal samples after heat treatment at 60 C for 5 minutes.

FIG. 8 demonstrates that ethanol reduces both anaerobic and aerobic bacterial species by several orders of magnitude in seconds.

FIG. 9 demonstrates the spore concentration of fecal donations from multiple donors over time.

FIG. 10 shows the strong correlation and linear correspondence between the measurement of DPA concentration by a coupled fluorescence assay and the viable spore colony forming units

FIG. 11 demonstrates the effect on various germination treatments on the ability to cultivate vegetative bacteria from a spore population.

FIG. 12 demonstrates the increase in bacterial diversity from using a germinant treatment to grow vegetative bacteria from spore populations.

FIG. 13 demonstrates the role of heat activation at various temperatures on spores from three different donor fecal samples.

FIG. 14 demonstrates a lysozyme treatment with heat activation improves germination at most temperatures.

FIG. 15 demonstrates spore concentrations present in a fecal sample grown on various medias.

FIG. 16 demonstrates similar spore production from incubating plates for 2 and 7 days after a spore population was germinated on plates with various medias.

FIG. 17 demonstrates the protective efficacy of the spore population in mice challenged with C. difficile as measured by the change in weight of mice over the course of the experiment. Each plot tracks the change in the individual mouse's weight relative to day −1 over the course of the experiment. The number of deaths over the course of the experiment is indicated at the top of the chart and demonstrated by a line termination prior to day 6. The top panels (from left to right) are the vehicle control arm, the fecal suspension arm, and the untreated naive control arm, while the bottom panels are the ethanol treated, gradient purified spore preparation; the ethanol treated, gradient purified, “germinable” spore preparation, and ethanol treated, gradient purified, “sporulatable” preparation.

FIG. 18 demonstrates the microbial diversity measured in the ethanol treated spore treatment sample and patient pre- and post-treatment samples. Total microbial diversity is defined using the Chao1 Alpha-Diversity Index and is measured at the same genomic sampling depths to confirm adequate sequence coverage to assay the microbiome in the target samples. The patient pretreatment (purple) harbored a microbiome that was significantly reduced in total diversity as compared to the ethanol treated spore treatment (red) and patient post treatment at days 5 (blue), 14 (orange), and 25 (green).

FIG. 19 demonstrates how the patient microbial ecology is shifted by treatment with an ethanol treated spore treatment from a dysbiotic state to a state of health. Principle coordinates analysis based on the total diversity and structure of the microbiome (Bray Curtis Beta Diversity) of the patient pre- and post-treatment delineates that the combination of engraftment of the OTUs from the spore treatment and the augmentation of the patient microbial ecology leads to a microbial ecology that is distinct from both the pretreatment microbiome and the ecology of the ethanol treated spore treatment.

FIG. 20 demonstrates the augmentation of bacteroides species in patients treated with the spore population. Comparing the number of Bacteroides colonies from fecal suspensions pre-treatment and in week 4 post treatment reveals an increase of 4 logs or greater. Colonies were enumerated by serial dilution and plating on Bacteroides Bile Esculin agar which is highly selective for the B. fragilis group. Species were determined by 16S full-length sequence identification.

FIG. 21 demonstrates the increase in number of species engrafting and species augmenting in patient's microbiomes after treatment with an ethanol-treated spore population. Relative abundance of species that engrafted or augmented as described were determined based on the number of 16S sequence reads. Each plot is from a different patient treated with the ethanol-treated spore population for recurrent C. difficile.

The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

DESCRIPTION OF THE TABLES

Table 1. List of Operational Taxonomic Units (OTU) with taxonomic assignments made to Genus, Species, and Phylogenetic Clade. Clade membership of bacterial OTUs is based on 16S sequence data. Clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood methods familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another, and (ii) within 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data, while OTUs falling within the same clade are closely related. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. Members of the same clade, due to their evolutionary relatedness, play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention. All OTUs are denoted as to their putative capacity to form spores and whether they are a Pathogen or Pathobiont (see Definitions for description of “Pathobiont”). NIAID Priority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or ‘Category-C’, and Opportunistic Pathogens are denoted as ‘OP’. OTUs that are not pathogenic or for which their ability to exist as a pathogen is unknown are denoted as ‘N’. The ‘SEQ ID Number’ denotes the identifier of the OTU in the Sequence Listing File and ‘Public DB Accession’ denotes the identifier of the OTU in a public sequence repository.

Table 2 contains bacterial OTUs identified from the 16s analysis of the ethanol treated spore population before and after a CsCl gradient purification.

Table 3 contains the mortality and weight change of mice treated with a donor fecal suspension and an ethanol and/or heat-treated spore preparation at various dilutions,

Table 4 contains OTUs identified from spore forming species generated by picking colonies from a spore preparation involving various heat treatments

Table 5 contains OTUs not identified in untreated fecal slurries, but identified in ethanol treated or heat treated spore populations.

Table 6 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor A.

Table 7 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor B.

Table 8 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor C.

Table 9 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor D.

Table 10 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor E.

Table 11 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor F.

Table 12 contains OTUs identified from growing ethanol treated spore populations on various media types.

Table 13. Species identified as “germinable” and “sporulatable” by colony picking approach

Table YYY. Species identified as “germinable” using 16S-V4 NGS approach.

Table ZZZ. Species identified as “sporulatable” using 16s-V4 NGS approach.

Table AC shows spore content data from 3 different ethanol treated spore preparations used to successfully treat 3 patients suffering from recurrent C. difficile infection.

Table AD. DPA doses in Table AC when normalized to 4×10⁵ SCFU per dose

Table GB. OTUs detected by a minimum of ten 16S-V4 sequence reads in at least a one ethanol treated spore preparation (pan-microbiome). OTUs that engraft in a treated patients and the percentage of patients in which they engraft are denoted, as are the clades, spore forming status, and Keystone OTU status. Starred OTUs occur in ≥80%, of the ethanol preps and engraft in ≥50%, of the treated patients.

Table GC ranks the top 20 OTUs by CES with the further requirement that an OTU must be shown to engraft to be a considered an element of a core ecology.

Table GD: Subsets of the Core Ecology tested in the C. difficile mouse model

Table GE: Results of bacterial compositions tested in a C. difficile mouse model.

Table GF. OTUs and their clade assignments tested in ternary combinations with results in the in vitro inhibition assay

Table ZA. Microbial compositions administered via oral gavage on Day −1

Table TAB. Population of OTUs on Days 2, 3 and 4 following dosing with Microbial Compositions

Table TAC. Population of Clades on Days 2, 3 and 4 following dosing with Microbial Compositions

Table TAD. Mortality by experimental group in mice challenged with 104.5 C. difficile spores on Day 0

DETAILED DESCRIPTION Overview

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety and efficacy.

It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.

Definitions

“Microbiota” refers to the community of microorganisms that occur (sustainably or transiently) in and on an animal subject, typically a mammal such as a human, including eukaryotes, archaea, bacteria, and viruses (including bacterial viruses i.e., phage).

“Microbiome” refers to the genetic content of the communities of microbes that live in and on the human body, both sustainably and transiently, including eukaryotes, archaea, bacteria, and viruses (including bacterial viruses (i.e., phage)), wherein “genetic content” includes genomic DNA, RNA such as ribosomal RNA, the epigenome, plasmids, and all other types of genetic information.

“Microbial Carriage” or simply “Carriage” refers to the population of microbes inhabiting a niche within or on humans. Carriage is often defined in terms of relative abundance. For example, OTU1 comprises 60% of the total microbial carriage, meaning that OTU1 has a relative abundance of 60% compared to the other OTUs in the sample from which the measurement was made. Carriage is most often based on genomic sequencing data where the relative abundance or carriage of a single OTU or group of OTUs is defined by the number of sequencing reads that are assigned to that OTU/s relative to the total number of sequencing reads for the sample.

“Microbial Augmentation” or simply “augmentation” refers to the establishment or significant increase of a population of microbes that are (i) absent or undetectable (as determined by the use of standard genomic and microbiological techniques) from the administered therapeutic microbial composition, (ii) absent, undetectable, or present at low frequencies in the host niche (as example: gastrointestinal tract, skin, anterior-nares, or vagina) before the delivery of the microbial composition, and (iii) are found after the administration of the microbial composition or significantly increase, for instance 2-fold, 5-fold, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or greater than 1×10⁸, in cases where they were present at low frequencies. The microbes that comprise an augmented ecology can be derived from exogenous sources such as food and the environment, or grow out from micro-niches within the host where they reside at low frequency.

The administration of the therapeutic microbial composition induces an environmental shift in the target niche that promotes favorable conditions for the growth of these commensal microbes. In the absence of treatment with a therapeutic microbial composition, the host can be constantly exposed to these microbes; however, sustained growth and the positive health effects associated with the stable population of increased levels of the microbes comprising the augmented ecology are not observed.

“Microbial Engraftment” or simply “engraftment” refers to the establishment of OTUs comprising a therapeutic microbial composition in a target niche that are absent in the treated host prior to treatment. The microbes that comprise the engrafted ecology are found in the therapeutic microbial composition and establish as constituents of the host microbial ecology upon treatment. Engrafted OTUs can establish for a transient period of time, or demonstrate long-term stability in the microbial ecology that populates the host post treatment with a therapeutic microbial composition. The engrafted ecology can induce an environmental shift in the target niche that promotes favorable conditions for the growth of commensal microbes capable of catalyzing a shift from a dysbiotic ecology to one representative of a health state.

“Ecological Niche” or simply “Niche” refers to the ecological space in which a an organism or group of organisms occupies. Niche describes how an organism or population or organisms responds to the distribution of resources, physical parameters (e.g., host tissue space) and competitors (e.g., by growing when resources are abundant, and when predators, parasites and pathogens are scarce) and how it in turn alters those same factors (e.g., limiting access to resources by other organisms, acting as a food source for predators and a consumer of prey).

“Dysbiosis” refers to a state of the microbiota of the gut or other body area in a subject, including mucosal or skin surfaces in which the normal diversity and/or function of the ecological network is disrupted. This unhealthy state can be due to a decrease in diversity, the overgrowth of one or more pathogens or pathobionts, symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject, or the shift to an ecological microbial network that no longer provides an essential function to the host subject, and therefore no longer promotes health.

“Pathobionts” or “Opportunistic Pathogens” refers to symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject.

“Phylogenetic tree” refers to a graphical representation of the evolutionary relationships of one genetic sequence to another that is generated using a defined set of phylogenetic reconstruction algorithms (e.g. parsimony, maximum likelihood, or Bayesian). Nodes in the tree represent distinct ancestral sequences and the confidence of any node is provided by a bootstrap or Bayesian posterior probability, which measures branch uncertainty.

“Operational taxonomic units,” “OTU” (or plural, “OTUs”) refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. In 16S embodiments, OTUs that share ≥97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU (see e.g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). In embodiments involving the complete genome, MLSTs, specific genes, or sets of genes OTUs that share ≥95%, average nucleotide identity are considered the same OTU (see e.g. Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Such characterization employs, e.g., WGS data or a whole genome sequence.

“Residual habitat products” refers to material derived from the habitat for microbiota within or on a human or animal. For example, microbiota live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community). Substantially free of residual habitat products means that the bacterial composition no longer contains the biological matter associated with the microbial environment on or in the human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community. Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms. Substantially free of residual habitat products may also mean that the bacterial composition contains no detectable cells from a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the bacterial composition contains no detectable viral (including bacterial viruses (i.e., phage)), fungal, mycoplasmal contaminants. In another embodiment, it means that fewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸ of the viable cells in the bacterial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting. Thus, contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology. Alternatively, reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10⁻⁸ or 10⁻⁹), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior. Other methods for confirming adequate purity include genetic analysis (e.g. PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.

“Clade” refers to the OTUs or members of a phylogenetic tree that are downstream of a statistically valid node in a phylogenetic tree. The clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity.

In microbiology, “16S sequencing” or “16S-rRNA” or “16S” refers to sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500 nucleotides in length and is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most bacteria.

The “V1-V9 regions” of the 16S rRNA refers to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature. Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU. A person of ordinary skill in the art can identify the specific hypervariable regions of a candidate 16S rRNA by comparing the candidate sequence in question to a reference sequence and identifying the hypervariable regions based on similarity to the reference hypervariable regions, or alternatively, one can employ Whole Genome Shotgun (WGS) sequence characterization of microbes or a microbial community.

The term “subject” refers to any animal subject including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), and household pets (e.g., dogs, cats, and rodents). The subject may be suffering from a dysbiosis, including, but not limited to, an infection due to a gastrointestinal pathogen or may be at risk of developing or transmitting to others an infection due to a gastrointestinal pathogen.

The term “phenotype” refers to a set of observable characteristics of an individual entity. As example an individual subject may have a phenotype of “health” or “disease”. Phenotypes describe the state of an entity and all entities within a phenotype share the same set of characteristics that describe the phenotype. The phenotype of an individual results in part, or in whole, from the interaction of the entities genome and/or microbiome with the environment.

The term “Network Ecology” refers to a consortium of OTUs that co-occur in some number of subjects. As used herein, a “network” is defined mathematically by a graph delineating how specific nodes (i.e. OTUs) and edges (connections between specific OTUs) relate to one another to define the structural ecology of a consortium of OTUs. Any given Network Ecology will possess inherent phylogenetic diversity and functional properties. A Network Ecology can also be defined in terms of function where for example the nodes would be comprised of elements such as, but not limited to, enzymes, clusters of orthologous groups (COGS; http://www.ncbi.nlm.nih.gov/books/NBK21090/), or KEGG pathways (www.genome.jp/kegg/).

The terms “Network Class”, “Core Network” and “Core Network Ecology” refer to a group of network ecologies that in general are computationally determined to comprise ecologies with similar phylogenetic and/or functional characteristics. A Core Network therefore contains important biological features, defined either phylogenetically or functionally, of a group (i.e., a cluster) of related network ecologies. One representation of a Core Network Ecology is a designed consortium of microbes, typically non-pathogenic bacteria, that represents core features of a set of phylogenetically or functionally related network ecologies seen in many different subjects. In many occurrences, a Core Network, while designed as described herein, exists as a Network Ecology observed in one or more subjects. Core Network ecologies are useful for reversing or reducing a dysbiosis in subjects where the underlying, related Network Ecology has been disrupted.

The term “Keystone OTU” refers to one or more OTUs that are common to many network ecologies and are members of networks ecologies that occur in many subjects (i.e. are pervasive) (FIG. 1). Due to the ubiquitous nature of Keystone OTUs, they are central to the function of network ecologies in healthy subjects and are often missing or at reduced levels in subjects with disease. Keystone OTUs may exist in low, moderate, or high abundance in subjects.

The term “non-Keystone OTU” refers to an OTU that is observed in a Network Ecology and is not a keystone OTU.

The term “Phylogenetic Diversity” refers to the biodiversity present in a given Network Ecology or Core Network Ecology based on the OTUs that comprise the network. Phylogenetic diversity is a relative term, meaning that a Network Ecology or Core Network that is comparatively more phylogenetically diverse than another network contains a greater number of unique species, genera, and taxonomic families. Uniqueness of a species, genera, or taxonomic family is generally defined using a phylogenetic tree that represents the genetic diversity all species, genera, or taxonomic families relative to one another. In another embodiment phylogenetic diversity may be measured using the total branch length or average branch length of a phylogenetic tree.

“Spore” or “endospore” refers to an entity, particularly a bacterial entity, which is in a dormant, non-vegetative and non-reproductive stage. Spores are generally resistant to environmental stress such as radiation, desiccation, enzymatic treatment, temperature variation, nutrient deprivation, and chemical disinfectants.

A “spore population” refers to a plurality of spores present in a composition. Synonymous terms used herein include spore composition, spore preparation, ethanol treated spore fraction and spore ecology. A spore population may be purified from a fecal donation, e.g. via ethanol or heat treatment, or a density gradient separation or any combination of methods described herein to increase the purity, potency and/or concentration of spores in a sample. Alternatively, a spore population may be derived through culture methods starting from isolated spore former species or spore former OTUs or from a mixture of such species, either in vegetative or spore form.

In one embodiment, the spore preparation comprises spore forming species wherein residual non-spore forming species have been inactivated by chemical or physical treatments including ethanol, detergent, heat, sonication, and the like; or wherein the non-spore forming species have been removed from the spore preparation by various separations steps including density gradients, centrifugation, filtration and/or chromatography; or wherein inactivation and separation methods are combined to make the spore preparation. In yet another embodiment, the spore preparation comprises spore forming species that are enriched over viable non-spore formers or vegetative forms of spore formers. In this embodiment, spores are enriched by 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared to all vegetative forms of bacteria. In yet another embodiment, the spores in the spore preparation undergo partial germination during processing and formulation such that the final composition comprises spores and vegetative bacteria derived from spore forming species.

A “germinant” is a material or composition or physical-chemical process capable of inducing vegetative growth of a bacterium that is in a dormant spore form, or group of bacteria in the spore form, either directly or indirectly in a host organism and/or in vitro.

A “sporulation induction agent” is a material or physical-chemical process that is capable of inducing sporulation in a bacterium, either directly or indirectly, in a host organism and/or in vitro.

To “increase production of bacterial spores” includes an activity or a sporulation induction agent. “Production” includes conversion of vegetative bacterial cells into spores and augmentation of the rate of such conversion, as well as decreasing the germination of bacteria in spore form, decreasing the rate of spore decay in vivo, or ex vivo, or to increasing the total output of spores (e.g. via an increase in volumetric output of fecal material).

The “colonization” of a host organism includes the non-transitory residence of a bacterium or other microscopic organism. As used herein, “reducing colonization” of a host subject's gastrointestinal tract (or any other microbiotal niche) by a pathogenic bacterium includes a reduction in the residence time of the pathogen in the gastrointestinal tract as well as a reduction in the number (or concentration) of the pathogen in the gastrointestinal tract or adhered to the luminal surface of the gastrointestinal tract. Measuring reductions of adherent pathogens may be demonstrated, e.g., by a biopsy sample, or reductions may be measured indirectly, e.g., by measuring the pathogenic burden in the stool of a mammalian host.

A “combination” of two or more bacteria includes the physical co-existence of the two bacteria, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the two bacteria.

A “cytotoxic” activity or bacterium includes the ability to kill a bacterial cell, such as a pathogenic bacterial cell. A “cytostatic” activity or bacterium includes the ability to inhibit, partially or fully, growth, metabolism, and/or proliferation of a bacterial cell, such as a pathogenic bacterial cell.

To be free of “non-comestible products” means that a bacterial composition or other material provided herein does not have a substantial amount of a non-comestible product, e.g., a product or material that is inedible, harmful or otherwise undesired in a product suitable for administration, e.g., oral administration, to a human subject. Non-comestible products are often found in preparations of bacteria from the prior art.

As used herein the term “vitamin” is understood to include any of various fat-soluble or water-soluble organic substances (non-limiting examples include vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folic acid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2 (i.e. MK-4, MK-7), folic acid and biotin) essential in minute amounts for normal growth and activity of the body and obtained naturally from plant and animal foods or synthetically made, pro-vitamins, derivatives, analogs.

As used herein, the term “minerals” is understood to include boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.

As used herein, the term “antioxidant” is understood to include any one or more of various substances such as beta-carotene (a vitamin A precursor), vitamin C, vitamin E, and selenium) that inhibit oxidation or reactions promoted by Reactive Oxygen Species (“ROS”) and other radical and non-radical species. Additionally, antioxidants are molecules capable of slowing or preventing the oxidation of other molecules. Non-limiting examples of antioxidants include astaxanthin, carotenoids, coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji (wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.

Compositions of the Invention

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety and efficacy.

It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.

Provided herein are therapeutic compositions containing a purified population of bacterial spores. As used herein, the terms “purify”, “purified” and “purifying” refer to the state of a population (e.g., a plurality of known or unknown amount and/or concentration) of desired bacterial spores, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacterial spore, or alternatively a removal or reduction of residual habitat products as described herein. In some embodiments, a purified population has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, a purified population has an amount and/or concentration of desired bacterial spores at or above an acceptable amount and/or concentration. In other embodiments, the ratio of desired-to-undesired activity (e.g. spores compared to vegetative bacteria), has changed by 2-, 5-, 10-, 30-, 100-, 300-, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, or greater than 1×10⁸. In other embodiments, the purified population of bacterial spores is enriched as compared to the starting material (e.g., a fecal material) from which the population is obtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.9999%, or greater than 99.999999% as compared to the starting material.

In certain embodiments, the purified populations of bacterial spores have reduced or undetectable levels of one or more pathogenic activities, such as toxicity, an ability to cause infection of the mammalian recipient subject, an undesired immunomodulatory activity, an autoimmune response, a metabolic response, or an inflammatory response or a neurological response. Such a reduction in a pathogenic activity may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material. In other embodiments, the purified populations of bacterial spores have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.

Provided are purified populations of bacterial spores that are substantially free of residual habitat products. In certain embodiments, this means that the bacterial spore composition no longer contains a substantial amount of the biological matter associated with the microbial community while living on or in the human or animal subject, and the purified population of spores may be 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contamination of the biological matter associated with the microbial community. Substantially free of residual habitat products may also mean that the bacterial spore composition contains no detectable cells from a human or animal, and that only microbial cells are detectable, in particular, only desired microbial cells are detectable. In another embodiment, it means that fewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸% of the cells in the bacterial composition are human or animal, as compared to microbial cells. In another embodiment, the residual habitat product present in the purified population is reduced at least a certain level from the fecal material obtained from the mammalian donor subject, e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%.

In one embodiment, substantially free of residual habitat products or substantially free of a detectable level of a pathogenic material means that the bacterial composition contains no detectable viral (including bacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmal contaminants, or a eukaryotic parasite such as a helminth. Alternatively, the purified spore populations are substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material. Alternatively, the purified spore population may processed by a method that kills, inactivates, or removes one or more specific undesirable viruses, such as an enteric virus, including norovirus, poliovirus or hepatitis A virus.

As described herein, purified spore populations can be demonstrated by genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, microscopic analysis, microbial analysis including germination and culturing, and methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial spores from non-desired, contaminating materials.

Exemplary biological materials include fecal materials such as feces or materials isolated from the various segments of the small and large intestines. Fecal materials are obtained from a mammalian donor subject, or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or from greater than 1000 donors, where such materials are then pooled prior to purification of the desired bacterial spores. In another embodiment, fecal materials can be obtained from a single donor subject over multiple times and pooled from multiple samples e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 32, 35, 40, 45, 48, 50, 100 samples from a single donor.

In alternative embodiments, the desired bacterial spores are purified from a single fecal material sample obtained from a single donor, and after such purification are combined with purified spore populations from other purifications, either from the same donor at a different time, or from one or more different donors, or both.

Mammalian donor subjects are generally of good health and have microbiota consistent with such good health. Often, the donor subjects have not been administered antibiotic compounds within a certain period prior to the collection of the fecal material. In certain embodiments, the donor subjects are not obese or overweight, and may have body mass index (BMI) scores of below 25, such as between 18.5 and 24.9. In other embodiments, the donor subjects are not mentally ill or have no history or familial history of mental illness, such as anxiety disorder, depression, bipolar disorder, autism spectrum disorders, schizophrenia, panic disorders, attention deficit (hyperactivity) disorders, eating disorders or mood disorders. In other embodiments, the donor subjects do not have irritable bowel disease (e.g., crohn's disease, ulcerative colitis), irritable bowel syndrome, celiac disease, colorectal cancer or a family history of these diseases. In other embodiments, donors have been screened for blood borne pathogens and fecal transmissible pathogens using standard techniques known to one in the art (e.g. nucleic acid testing, serological testing, antigen testing, culturing techniques, enzymatic assays, assays of cell free fecal filtrates looking for toxins on susceptible cell culture substrates).

In some embodiments, donors are also selected for the presence of certain genera and/or species that provide increased efficacy of therapeutic compositions containing these genera or species. In other embodiments, donors are preferred that produce relatively higher concentrations of spores in fecal material than other donors. In further embodiments, donors are preferred that provide fecal material from which spores having increased efficacy are purified; this increased efficacy is measured using in vitro or in animal studies as described below. In some embodiments, the donor may be subjected to one or more pre-donation treatments in order to reduce undesired material in the fecal material, and/or increase desired spore populations.

It is advantageous to screen the health of the donor subject prior to and optionally, one or more times after, the collection of the fecal material. Such screening identifies donors carrying pathogenic materials such as viruses (HIV, hepatitis, polio) and pathogenic bacteria. Post-collection, donors are screened about one week, two weeks, three weeks, one month, two months, three months, six months, one year or more than one year, and the frequency of such screening may be daily, weekly, bi-weekly, monthly, bi-monthly, semi-yearly or yearly. Donors that are screened and do not test positive, either before or after donation or both, are considered “validated” donors.

Solvent treatments. To purify the bacterial spores, the fecal material is subjected to one or more solvent treatments. A solvent treatment is a miscible solvent treatment (either partially miscible or fully miscible) or an immiscible solvent treatment. Miscibility is the ability of two liquids to mix with each to form a homogeneous solution. Water and ethanol, for example, are fully miscible such that a mixture containing water and ethanol in any ratio will show only one phase. Miscibility is provided as a wt/wt %, or weight of one solvent in 100 g of final solution. If two solvents are fully miscible in all proportions, their miscibility is 100%. Provided as fully miscible solutions with water are alcohols, e.g., methanol, ethanol, isopropanol, butanol, propanediol, butanediol, etc. The alcohols can be provided already combined with water; e.g., a solution containing 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% or greater than 95%. Other solvents are only partially miscible, meaning that only some portion will dissolve in water. Diethyl ether, for example, is partially miscible with water. Up to 7 grams of diethyl ether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. If more diethyl ether is added, a two-phase solution will result with a distinct diethyl ether layer above the water. Other partially miscible materials include ethers, propanoate, butanoate, chloroform, dimethoxyethane, or tetrahydrofuran. In contrast, an oil such as an alkane and water are immiscible and form two phases. Further, immiscible treatments are optionally combined with a detergent, either an ionic detergent or a non-ionic detergent. Exemplary detergents include Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or a polyol. The solvent treatment steps reduces the viability of non-spore forming bacterial species by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and it may optionally reduce the viability of contaminating protists, parasites and/or viruses.

Chromatography treatments. To purify spore populations, the fecal materials are subjected to one or more chromatographic treatments, either sequentially or in parallel. In a chromatographic treatment, a solution containing the fecal material is contacted with a solid medium containing a hydrophobic interaction chromatographic (HIC) medium or an affinity chromatographic medium. In an alternative embodiment, a solid medium capable of absorbing a residual habitat product present in the fecal material is contacted with a solid medium that adsorbs a residual habitat product. In certain embodiments, the HIC medium contains sepharose or a derivatized sepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, or butyl-s sepharose. In other embodiments, the affinity chromatographic medium contains material derivatized with mucin type I, II, III, IV, V, or VI, or oligosaccharides derived from or similar to those of mucins type I, II, III, IV, V, or VI. Alternatively, the affinity chromatographic medium contains material derivatized with antibodies that recognize spore-forming bacteria.

Mechanical treatments. Provided herein is the physical disruption of the fecal material, particularly by one or more mechanical treatment such as blending, mixing, shaking, vortexing, impact pulverization, and sonication. As provided herein, the mechanical disrupting treatment substantially disrupts a non-spore material present in the fecal material and does not substantially disrupt a spore present in the fecal material, or it may disrupt the spore material less than the non-spore material, e.g. 2-fold less, 5-, 10-, 30-, 100-, 300-, 1000- or greater than 1000-fold less. Furthermore, mechanical treatment homogenizes the material for subsequent sampling, testing, and processing. Mechanical treatments optionally include filtration treatments, where the desired spore populations are retained on a filter while the undesirable (non-spore) fecal components to pass through, and the spore fraction is then recovered from the filter medium. Alternatively, undesirable particulates and eukaryotic cells may be retained on a filter while bacterial cells including spores pass through. In some embodiments the spore fraction retained on the filter medium is subjected to a diafiltration step, wherein the retained spores are contacted with a wash liquid, typically a sterile saline-containing solution or other diluent such as a water compatible polymer including a low-molecular polyethylene glycol (PEG) solution, in order to further reduce or remove the undesirable fecal components.

Thermal treatments. Provided herein is the thermal disruption of the fecal material. Generally, the fecal material is mixed in a saline-containing solution such as phosphate-buffered saline (PBS) and subjected to a heated environment, such as a warm room, incubator, water-bath, or the like, such that efficient heat transfer occurs between the heated environment and the fecal material. Preferably the fecal material solution is mixed during the incubation to enhance thermal conductivity and disrupt particulate aggregates. Thermal treatments can be modulated by the temperature of the environment and/or the duration of the thermal treatment. For example, the fecal material or a liquid comprising the fecal material is subjected to a heated environment, e.g., a hot water bath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at least about 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours. In certain embodiments the thermal treatment occurs at two different temperatures, such as 30 seconds in a 100 degree Celsius environment followed by 10 minutes in a 50 degree Celsius environment. In preferred embodiments the temperature and duration of the thermal treatment are sufficient to kill or remove pathogenic materials while not substantially damaging or reducing the germination-competency of the spores. In other preferred embodiments, the temperature and duration of the thermal treatment is short enough to reduce the germination of the spore population.

Irradiation treatments. Provided are methods of treating the fecal material or separated contents of the fecal material with ionizing radiation, typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations. For example, ultraviolet radiation at 254 nm provided at an energy level below about 22,000 microwatt seconds per cm² will not generally destroy desired spores.

Centrifugation and density separation treatments. Provided are methods of separating desired spore populations from the other components of the fecal material by centrifugation. A solution containing the fecal material is subjected to one or more centrifugation treatments, e.g., at about 200×g, 1000×g, 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g. Differential centrifugation separates desired spores from undesired non-spore material; at low forces the spores are retained in solution, while at higher forces the spores are pelleted while smaller impurities (e.g., virus particles, phage, microscopic fibers, biological macromolecules such as free protein, nucleic acids and lipids) are retained in solution. For example, a first low force centrifugation pellets fibrous materials; a second, higher force centrifugation pellets undesired eukaryotic cells, and a third, still higher force centrifugation pellets the desired spores while smaller contaminants remain in suspension. In some embodiments density or mobility gradients or cushions (e.g., step cushions), such as CsCl, Percoll, Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separate desired spore populations from other materials in the fecal material.

Also provided herein are methods of producing spore populations that combine two or more of the treatments described herein in order to synergistically purify the desired spores while killing or removing undesired materials and/or activities from the spore population. It is generally desirable to retain the spore populations under non-germinating and non-growth promoting conditions and media, in order to minimize the growth of pathogenic bacteria present in the spore populations and to minimize the germination of spores into vegetative bacterial cells.

Purified spore populations. As described herein, purified spore populations contain combinations of commensal bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to a mammalian subject. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, Fusobacterium spp., Klebsiella spp. and vancomycin-resistant Enterococcus spp., so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens. In one embodiment, the purified spore populations can engraft in the host and remain present for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 25 days, 30 days, 60 days, 90 days, or longer than 90 days. Additionally, the purified spore populations can induce other healthy commensal bacteria found in a healthy gut to engraft in the host that are not present in the purified spore populations or present at lesser levels and therefore these species are considered to “augment” the delivered spore populations. In this manner, commensal species augmentation of the purified spore population in the recipient's gut leads to a more diverse population of gut microbiota then present initially.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio, Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter, Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides, Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia, Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales, Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus, Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium, Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix, Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium, Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium, Gemmiger, Geobacillus, Gloeobacter, Holdemania, Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira, Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora, Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter, Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor, Robinsoniella, Roseburia, Rum inococcaceae, Ruminococcus, Saccharomonospora, Sarcina, Solobacterium, Sporobacter, Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella, Syntrophococcus, Thermoanaerobacter, Thermobifida, Turicibacter

Preferred bacterial species are provided at Table 1 and demarcated as spore formers. Where specific strains of a species are provided, one of skill in the art will recognize that other strains of the species can be substituted for the named strain.

In some embodiments, spore-forming bacteria are identified by the presence of nucleic acid sequences that modulate sporulation. In particular, signature sporulation genes are highly conserved across members of distantly related genera including Clostridium and Bacillus. Traditional approaches of forward genetics have identified many, if not all, genes that are essential for sporulation (spo). The developmental program of sporulation is governed in part by the successive action of four compartment-specific sigma factors (appearing in the order σF, σE, σG and σK), whose activities are confined to the forespore (σF and σG) or the mother cell (σE and σK). In other embodiments, spore-forming bacteria are identified by the biochemical activity of DPA producing enzymes or by analyzing DPA content of cultures. As part of the bacterial sporulation, large amounts of DPA are produced, and comprise 5-15% of the mass of a spore. Because not all viable spores germinate and grow under known media conditions, it is difficult to assess a total spore count in a population of bacteria. As such, a measurement of DPA content highly correlates with spore content and is an appropriate measure for characterizing total spore content in a bacterial population.

[Provided are spore populations containing more than one type of bacterium. As used herein, a “type” or more than one “types” of bacteria may be differentiated at the genus level, the species, level, the sub-species level, the strain level or by any other taxonomic method, as described herein and otherwise known in the art.

In some embodiments all or essentially all of the bacterial spores present in a purified population are obtained from a fecal material treated as described herein or otherwise known in the art. In alternative embodiments, one or more than one bacterial spores or types of bacterial spores are generated in culture and combined to form a purified spore population. In other alternative embodiments, one or more of these culture-generated spore populations are combined with a fecal material-derived spore population to generate a hybrid spore population. Bacterial compositions may contain at least two types of these preferred bacteria, including strains of the same species. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 or more than 20 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species.

Thus, provided herein are methods for production of a composition containing a population of bacterial spores suitable for therapeutic administration to a mammalian subject in need thereof. And the composition is produced by generally following the steps of: (a) providing a fecal material obtained from a mammalian donor subject; and (b) subjecting the fecal material to at least one purification treatment or step under conditions such that a population of bacterial spores is produced from the fecal material. The composition is formulated such that a single oral dose contains at least about 1×10⁴ colony forming units of the bacterial spores, and a single oral dose will typically contain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ CFUs of the bacterial spores. The presence and/or concentration of a given type of bacterial spore may be known or unknown in a given purified spore population. If known, for example the concentration of spores of a given strain, or the aggregate of all strains, is e.g., 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³ 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ viable bacterial spores per gram of composition or per administered dose.

In some formulations, the composition contains at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a mass basis. In some formulations, the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacterial spore compositions are generally formulated for oral or gastric administration, typically to a mammalian subject. In particular embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge. In some embodiments, such formulations contain or are coated by an enteric coating to protect the bacteria through the stomach and small intestine, although spores are generally resistant to the stomach and small intestines. In other embodiments, the bacterial spore compositions may be formulated with a germinant to enhance engraftment, or efficacy. In yet other embodiments, the bacterial spore compositions may be co-formulated or co-administered with prebiotic substances, to enhance engraftment or efficacy.

The bacterial spore compositions may be formulated to be effective in a given mammalian subject in a single administration or over multiple administrations. For example, a single administration is substantially effective to reduce Cl. difficile and/or Cl. difficile toxin content in a mammalian subject to whom the composition is administered. Substantially effective means that Cl. difficile and/or Cl. difficile toxin content in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% following administration of the composition. Alternatively, efficacy may be measured by the absence of diarrheal symptoms or the absence of carriage of C. difficile or C. difficile toxin after 2 day, 4 days, 1 week, 2 weeks, 4 weeks, 8 weeks or longer than 8 weeks.

Bacterial Compositions

Provided are bacteria and combinations of bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to mammalian hosts. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth, proliferation, and/or colonization of one or a plurality of pathogenic bacteria in the dysbiotic microbiotal niche, so that a healthy, diverse and protective microbiota colonizes and populates the intestinal lumen to establish or reestablish ecological control over pathogens or potential pathogens (e.g., some bacteria are pathogenic bacteria only when present in a dysbiotic environment). Inhibition of pathogens includes those pathogens such as C. difficile, Salmonella spp., enteropathogenic E. coli, multi-drug resistant bacteria such as Klebsiella, and E. coli, Carbapenem-resistent Enterobacteriaceae (CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant Enterococci (VRE).

As used herein, a “type” or more than one “types” of bacteria may be differentiated at the genus level, the species, level, the sub-species level, the strain level or by any other taxonomic method, as described herein and otherwise known in the art.

Bacterial compositions may comprise two types of bacteria (termed “binary combinations” or “binary pairs”) or greater than two types of bacteria. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at least 40, at least 50 or greater than 50 types of bacteria, as defined by species or operational taxonomic unit (OTU), or otherwise as provided herein.

In another embodiment, the number of types of bacteria present in a bacterial composition is at or below a known value. For example, in such embodiments the bacterial composition comprises 50 or fewer types of bacteria, such as 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer, or 9 or fewer types of bacteria, 8 or fewer types of bacteria, 7 or fewer types of bacteria, 6 or fewer types of bacteria, 5 or fewer types of bacteria, 4 or fewer types of bacteria, or 3 or fewer types of bacteria. In another embodiment, a bacterial composition comprises from 2 to no more than 40, from 2 to no more than 30, from 2 to no more than 20, from 2 to no more than 15, from 2 to no more than 10, or from 2 to no more than 5 types of bacteria.

Bacterial Compositions Described by Species

Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus). In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus). In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In another embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Acidaminococcus intestinalis, Bacteroides ovatus, two strains of Bifidobacterium adolescentis, two strains of Bifidobacterium longum, Blautia producta, Clostridium cocleatum, Collinsella aerofaciens, two strains of Dorea longicatena, Escherichia coli, Eubacterium desmolans, Eubacterium eligens, Eubacterium limosum, four strains of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lachnospira pectinoshiza, Lactobacillus casei, Lactobacillus casei/paracasei, Paracateroides distasonis, Raoultella sp., one strain of Roseburia (chosen from Roseburia faecalis or Roseburia faecis), Roseburia intestinalis, two strains of Ruminococcus torques, two strains of Ruminococcus obeum, and Streptococcus mitis. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In yet another embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bamesiella intestinihominis; Lactobacillus reuteri; a species characterized as one of Enterococcus hirae, Enterococus faecium, or Enterococcus durans; a species characterized as one of Anaerostipes caccae or Clostridium indolis; a species characterized as one of Staphylococcus warneri or Staphylococcus pasteuri; and Adlercreutzia equolifaciens. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In other embodiments, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bartlettii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniform is, three strains of Escherichia coli, and Lactobacillus sp. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss. Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III, Blautia producta (previously known as Peptostreptococcus productus I), Ruminococcus bronii, Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacterium rectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroides capillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacterium haffii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus cailidus, Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilis ss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum, Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1, Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum, Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT, Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacterium nucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcus morbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH, -CC; Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H, -AG-M, -AJ, -BN-1; Bacteroides clostridliformis ss. clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroides ruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus, Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, and Succinivibrio A. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.

Bacterial Compositions Described by Operational Taxonomic Unit (OTUs)

Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1.

In one embodiment, the OTUs can be characterized by one or more of the variable regions of the 16S sequence (V1-V9). These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature. (See, e.g., Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978)). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU.

Bacterial Compositions Exclusive of Certain Bacterial Species or Strains

In one embodiment, the bacterial composition does not comprise at least one of Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise at least one of Acidaminococcus intestinalis, Bacteroides ovatus, two species of Bifidobacterium adolescentis, two species of Bifidobacterium longum, Collinsella aerofaciens, two species of Dorea longicatena, Escherichia coli, Eubacterium eligens, Eubacterium limosum, four species of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lactobacillus casei, Lactobacillus paracasei, Paracateroides distasonis, Raoultella sp., one species of Roseburia (chosen from Roseburia faecalis or Roseburia faecis), Roseburia intestinalis, two species of Ruminococcus torques, and Streptococcus mitis.

In yet another embodiment, the bacterial composition does not comprise at least one of Barnesiella intestinihominis; Lactobacillus reuteri; a species characterized as one of Enterococcus hirae, Enterococus faecium, or Enterococcus durans; a species characterized as one of Anaerostipes caccae or Clostridium indolis; a species characterized as one of Staphylococcus warneri or Staphylococcus pasteuri; and Adlercreutzia equolifaciens.

In other embodiments, the bacterial composition does not comprise at least one of Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum.

In another embodiment, the bacterial composition does not comprise at least one of Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains of Escherichia coli, and Lactobacillus sp.

In another embodiment, the bacterial composition does not comprise at least one of Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta (previously known as Peptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise at least one of Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum.

In another embodiment, the bacterial composition does not comprise at least one of more than one Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum.

In another embodiment, the bacterial composition does not comprise at least one of Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus.

In another embodiment, the bacterial composition does not comprise at least one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus.

In another embodiment, the bacterial composition does not comprise at least one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss. Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III, Blautia producta (previously known as Peptostreptococcus productus I), Ruminococcus bromii, Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacterium rectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroides capillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacterium hallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus cailidus, Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilis ss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum, Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1, Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum, Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT, Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacterium nucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcus morbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH, -CC; Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H, -AG-M, -AJ, -BN-1; Bacteroides clostridiiformis ss. clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroides ruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus, Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, and Succinivibrio A.

Inhibition of Bacterial Pathogens

In some embodiments, the bacterial composition provides a protective or therapeutic effect against infection by one or more GI pathogens of interest.

A list of exemplary bacterial pathogens is provided in Table 1 as indicated by pathogen status.

In some embodiments, the pathogenic bacterium is selected from the group consisting of Yersinia, Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug resistant bacteria, extended spectrum beta-lactam resistant Enterococci (ESBL), Carbapenem-resistent Enterobacteriaceae (CRE), and vancomycin-resistant Enterococci (VRE).

In some embodiments, these pathogens include, but are not limited to, Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli (such as, but not limited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacter pylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the pathogen of interest is at least one pathogen chosen from Clostridium difficile, Salmonella spp., pathogenic Escherichia coli, vancomycin-resistant Enterococcus spp., and extended spectrum beta-lactam resistant Enterococci (ESBL).

Purified Spore Populations

In some embodiments, the bacterial compositions comprise purified spore populations. Purified spore populations contain combinations of commensal bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to a mammalian subject. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, and vancomycin-resistant Enterococcus spp., so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens. In some embodiments, yeast spores and other fungal spores are also purified and selected for therapeutic use.

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety.

It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.

Provided herein are therapeutic compositions containing a purified population of bacterial spores. As used herein, the terms “purify”, “purified” and “purifying” refer to the state of a population (e.g., a plurality of known or unknown amount and/or concentration) of desired bacterial spores, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacterial spore, or alternatively a removal or reduction of residual habitat products as described herein. In some embodiments, a purified population has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, a purified population has an amount and/or concentration of desired bacterial spores at or above an acceptable amount and/or concentration. In other embodiments, the purified population of bacterial spores is enriched as compared to the starting material (e.g., a fecal material) from which the population is obtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material.

In certain embodiments, the purified populations of bacterial spores have reduced or undetectable levels of one or more pathogenic activities, such as toxicity, an infection of the mammalian recipient subject, an immunomodulatory activity, an autoimmune response, a metabolic response, or an inflammatory response or a neurological response. Such a reduction in a pathogenic activity may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material. In other embodiments, the purified populations of bacterial spores have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.

Provided are purified populations of bacterial spores that are substantially free of residual habitat products. In certain embodiments, this means that the bacterial spore composition no longer contains a substantial amount of the biological matter associated with the microbial community while living on or in the human or animal subject, and the purified population of spores may be 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contamination of the biological matter associated with the microbial community. Substantially free of residual habitat products may also mean that the bacterial spore composition contains no detectable cells from a human or animal, and that only microbial cells are detectable, in particular, only desired microbial cells are detectable. In another embodiment, it means that fewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸% of the cells in the bacterial composition are human or animal, as compared to microbial cells. In another embodiment, the residual habitat product present in the purified population is reduced at least a certain level from the fecal material obtained from the mammalian donor subject, e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%.

In one embodiment, substantially free of residual habitat products or substantially free of a detectable level of a pathogenic material means that the bacterial composition contains no detectable viral (including bacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmal contaminants, or a eukaryotic parasite such as a helminth. Alternatively, the purified spore populations are substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material.

As described herein, purified spore populations can be demonstrated by genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial spores from non-desired, contaminating materials.

Exemplary biological materials include fecal materials such as feces or materials isolated from the various segments of the small and large intestines. Fecal materials are obtained from a mammalian donor subject, or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or from greater than 1000 donors, where such materials are then pooled prior to purification of the desired bacterial spores.

In alternative embodiments, the desired bacterial spores are purified from a single fecal material sample obtained from a single donor, and after such purification are combined with purified spore populations from other purifications, either from the same donor at a different time, or from one or more different donors, or both.

Preferred bacterial genera include Acetonema, Alkaliphilus, Alicyclobacillus, Amphibacillus, Ammonifex, Anaerobacter, Anaerofustis, Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Blautia, Brevibacillus, Bryantella, Caldicellulosiruptor, Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Clostridium, Cohnella, Coprococcus, Dendrosporobacter Desulfitobacterium, Desulfosporosinus, Desulfotomaculum, Dorea, Eubacterium, Faecalibacterium, Filifactor, Geobacillus, Halobacteroides, Heliobacillus, Heliobacterium, Heliophilum, Heliorestis, Lachnoanaerobaculum, Lysinibacillus, Moorella, Oceanobacillus, Orenia (S.), Oxalophagus, Oxobacter, Paenibacillus, Pelospora, Pelotomaculum, Propionispora, Roseburia, Ruminococcus, Sarcina, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Subdoligranulum, Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus, Thermoanaerobacter, and Thermosinus.

Preferred bacterial species are provided at Table X4. Where specific strains of a species are provided, one of skill in the art will recognize that other strains of the species can be substituted for the named strain.

In some embodiments, spore-forming bacteria are identified by the presence of nucleic acid sequences that modulate sporulation. In particular, signature sporulation genes are highly conserved across members of distantly related genera including Clostridium and Bacillus. Traditional approaches of forward genetics have identified many, if not all, genes that are essential for sporulation (spo). The developmental program of sporulation is governed in part by the successive action of four compartment-specific sigma factors (appearing in the order of, σE, σG and σK), whose activities are confined to the forespore (σF and σG) or the mother cell (σE and σK).

Provided are spore populations containing more than one type of bacterium. As used herein, a “type” or more than one “types” of bacteria may be differentiated at the genus level, the species, level, the sub-species level, the strain level or by any other taxonomic method, as described herein and otherwise known in the art.

In some embodiments, all or essentially all of the bacterial spores present in a purified population are obtained from a fecal material treated as described herein or otherwise known in the art. In alternative embodiments, one or more than one bacterial spores or types of bacterial spores are generated in culture and combined to form a purified spore population. In other alternative embodiments, one or more of these culture-generated spore populations are combined with a fecal material-derived spore population to generate a hybrid spore population. Bacterial compositions may contain at least two types of these preferred bacteria, including strains of the same species. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 or more than 20 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species.

Thus, provided herein are methods for production of a composition containing a population of bacterial spores suitable for therapeutic administration to a mammalian subject in need thereof. And the composition is produced by generally following the steps of: (a) providing a fecal material obtained from a mammalian donor subject; and (b) subjecting the fecal material to at least one purification treatment or step under conditions such that a population of bacterial spores is produced from the fecal material. The composition is formulated such that a single oral dose contains at least about 1×10⁴ colony forming units of the bacterial spores, and a single oral dose will typically contain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ CFUs of the bacterial spores. The presence and/or concentration of a given type of bacteria spore may be known or unknown in a given purified spore population. If known, for example the concentration of spores of a given strain, or the aggregate of all strains, is e.g., 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ viable bacterial spores per gram of composition or per administered dose.

In some formulations, the composition contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a mass basis. In some formulations, the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacterial spore compositions are generally formulated for oral or gastric administration, typically to a mammalian subject. In particular embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge. In some embodiments, such formulations contain or are coated by an enteric coating to protect the bacteria through the stomach and small intestine, although spores are generally resistant to the stomach and small intestines.

The bacterial spore compositions may be formulated to be effective in a given mammalian subject in a single administration or over multiple administrations. For example, a single administration is substantially effective to reduce Cl. difficile and/or Cl. difficile toxin content in a mammalian subject to whom the composition is administered. Substantially effective means that Cl. difficile and/or Cl. difficile toxin content in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% following administration of the composition.

METHODS OF THE INVENTION

Methods for Determining 16S Sequences

OTUs can be defined either by full 16S sequencing of the rRNA gene, by sequencing of a specific hypervariable region of this gene (i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination of hypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial 16S rDNA is approximately 1500 nucleotides in length and is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most microbes.

Using well known techniques, in order to determine the full 16S sequence or the sequence of any hypervariable region of the 16S sequence, genomic DNA is extracted from a bacterial sample, the 16S rDNA (full region or specific hypervariable regions) amplified using polymerase chain reaction (PCR), the PCR products cleaned, and nucleotide sequences delineated to determine the genetic composition of 16S gene or subdomain of the gene. If full 16S sequencing is performed, the sequencing method used may be, but is not limited to, Sanger sequencing. If one or more hypervariable regions are used, such as the V4 region, the sequencing can be, but is not limited to being, performed using the Sanger method or using a next-generation sequencing method, such as an IIlumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.

OTUs can be defined by a combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof, full-genome sequence, or partial genome sequence generated using amplified genetic products, or whole genome sequence (WGS). Using well defined methods DNA extracted from a bacterial sample will have specific genomic regions amplified using PCR and sequenced to determine the nucleotide sequence of the amplified products. In the whole genome shotgun (WGS) method, extracted DNA will be directly sequenced without amplification. Sequence data can be generated using any sequencing technology including, but not limited to Sanger, IIlumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences, and/or Oxford Nanopore.

Methods for Preparing a Bacterial Composition for Administration to a Subject

Methods for producing bacterial compositions can include three main processing steps, combined with one or more mixing steps. The steps include organism banking, organism production, and preservation.

For banking, the strains included in the bacterial composition may be (1) isolated directly from a specimen or taken from a banked stock, (2) optionally cultured on a nutrient agar or broth that supports growth to generate viable biomass, and (3) the biomass optionally preserved in multiple aliquots in long-term storage.

In embodiments that use a culturing step, the agar or broth can contain nutrients that provide essential elements and specific factors that enable growth. An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione. A variety of microbiological media and variations are well known in the art (e.g. R. M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Medium can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture. The strains in the bacterial composition may be cultivated alone, as a subset of the bacterial composition, or as an entire collection comprising the bacterial composition. As an example, a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for a time sufficient to build biomass. For bacterial compositions for human use, this is often at 37° C. temperature, pH, and other parameter with values similar to the normal human niche. The environment can be actively controlled, passively controlled (e.g., via buffers), or allowed to drift. For example, for anaerobic bacterial compositions (e.g., gut microbiota), an anoxic/reducing environment can be employed. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen. As an example, a culture of a bacterial composition can be grown at 37° C., pH 7, in the medium above, pre-reduced with 1 g/L cysteine□HCl.

When the culture has generated sufficient biomass, it can be preserved for banking. The organisms can be placed into a chemical milieu that protects from freezing (adding ‘cryoprotectants’), drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation. Containers are generally impermeable and have closures that assure isolation from the environment. Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below −80° C.). Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term bacterial composition storage stability at temperatures elevated above cryogenic. If the bacterial composition comprises spore forming species and results in the production of spores, the final composition can be purified by additional means, such as density gradient centrifugation preserved using the techniques described above. Bacterial composition banking can be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of cryopreservation, a bacterial composition culture can be harvested by centrifugation to pellet the cells from the culture medium, the supernate decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C. for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.

Organism production can be conducted using similar culture steps to banking, including medium composition and culture conditions. It can be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there can be several subcultivations of the bacterial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the bacterial composition and renders it acceptable for administration via the chosen route. For example, a bacterial composition can be cultivated to a concentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium can be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer. The suspension can then be freeze-dried to a powder and titrated.

After drying, the powder can be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.

Administration of Bacterial Compositions.

The bacterial compositions of the invention are suitable for administration to mammals and non-mammalian animals in need thereof. In certain embodiments, the mammalian subject is a human subject who has one or more symptoms of a dysbiosis, including but not limited to overgrowth of an undesired pathobiont or pathogen, reduced representation of key bacterial taxa such as the Bacteroidetes or Firmicutes or genera or species thereof, or reduced diversity of microbial species compared to a healthy individual, or reduced overall abundance of anaerobic bacteria.

When the mammalian subject is suffering from a disease, disorder or condition characterized by an aberrant microbiota, the bacterial compositions described herein are suitable for treatment thereof. In some embodiments, the mammalian subject has not received antibiotics in advance of treatment with the bacterial compositions. For example, the mammalian subject has not been administered at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has received one or more treatments with one or more different antibiotic compounds and such treatment(s) resulted in no improvement or a worsening of symptoms. In some embodiments, the spore composition is administered following a successful course of antibiotics to prevent dysbiosis and enhance recovery of a diverse, healthy microbiota.

In some embodiments, the gastrointestinal disease, disorder or condition is diarrhea caused by C. difficile including recurrent C. difficile infection, ulcerative colitis, colitis, Crohn's disease, or irritable bowel disease. Beneficially, the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition. In some embodiments the therapeutic composition is administered at intervals greater than two days, such as once every three, four, five or six days, or every week or less frequently than every week. Or the preparation may be administered intermittently according to a set schedule, e.g., once a day, once weekly, or once monthly, or when the subject relapses from the primary illness. In another embodiment, the preparation may be administered on a long-term basis to individuals who are at risk for infection with or who may be carriers of these pathogens, including individuals who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other healthcare professionals).

Also provided are methods of treating or preventing a mammalian subject suffering from or at risk of developing a metabolic disease, and disorder or condition selected from the group consisting of diabetes, metabolic syndrome, obesity, heart disease, autoimmune disease, liver disease, and autism using the therapeutic compositions provided herein.

In embodiments, the bacterial spore composition is administered enterically. This preferentially includes oral administration, or by an oral or nasal tube (including nasogastric, nasojejunal, oral gastric, or oral jejunal). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy). The bacterial composition may be administered to at least one region of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum. In some embodiments, it is administered to all regions of the gastrointestinal tract. The bacterial compositions may be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids. The bacterial compositions may also be administered in gel or liquid form by the oral route or through a nasogastric tube, or by the rectal route in a gel or liquid form, by enema or instillation through a colonoscope or by a suppository.

If the composition is administered colonoscopically and, optionally, if the bacterial composition is administered by other rectal routes (such as an enema or suppository) or even if the subject has an oral administration, the subject may have a colonic-cleansing preparation. The colon-cleansing preparation can facilitate proper use of the colonoscope or other administration devices, but even when it does not serve a mechanical purpose it can also maximize the proportion of the bacterial composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. Any ordinarily acceptable colonic-cleansing preparation may be used such as those typically provided when a subject undergoes a colonoscopy.

To evaluate the subject, symptoms of dysbiosis are evaluated post treatment ranging from 1 day to 6 months after administration of the purified spore population. Fecal material is collected during this period and the microbes present in the gastrointestinal tract can be assessed by 16S rDNA or metagenomic sequencing analysis or other analyses commonly used by the skilled artisan. Repopulation by species provided by the spore population as well as Augmentation by commensal microbes not present in the spore population will occur in this time as the spore population catalyzes a reshaping of the gut ecology to a state of healthy biosis. The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments and should not be construed to limit the scope. The skilled artisan readily recognizes that many other embodiments are encompassed. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art.

Methods of Treating a Subject

In some embodiments, the compositions disclosed herein are administered to a patient or a user (sometimes collectively referred to as a “subject”). As used herein “administer” and “administration” encompasses embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose, and also situations in which a user uses a bacteria composition in a certain manner and/or for a certain purpose independently of or in variance to any instructions received from a second person. Non-limiting examples of embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose include when a physician prescribes a course of conduct and/or treatment to a patient, when a parent commands a minor user (such as a child) to consume a bacterial composition, when a trainer advises a user (such as an athlete) to follow a particular course of conduct and/or treatment, and when a manufacturer, distributer, or marketer recommends conditions of use to an end user, for example through advertisements or labeling on packaging or on other materials provided in association with the sale or marketing of a product.

The bacterial compositions offer a protective and/or therapeutic effect against infection by one or more GI pathogens of interest and can be administered after an acute case of infection has been resolved in order to prevent relapse, during an acute case of infection as a complement to antibiotic therapy if the bacterial composition is not sensitive to the same antibiotics as the GI pathogen, or to prevent infection or reduce transmission from disease carriers.

The present bacterial compositions can be useful in a variety of clinical situations. For example, the bacterial compositions can be administered as a complementary treatment to antibiotics when a patient is suffering from an acute infection, to reduce the risk of recurrence after an acute infection has subsided, or when a patient will be in close proximity to others with or at risk of serious gastrointestinal infections (physicians, nurses, hospital workers, family members of those who are ill or hospitalized).

The present bacterial compositions can be administered to animals, including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), and household pets (e.g., dogs, cats, rodents).

In the present method, the bacterial composition can be administered enterically, in other words, by a route of access to the gastrointestinal tract. This includes oral administration, rectal administration (including enema, suppository, or colonoscopy), by an oral or nasal tube (nasogastric, nasojejunal, oral gastric, or oral jejunal), as detailed more fully herein.

Pretreatment Protocols

Prior to administration of the bacterial composition, the patient can optionally have a pretreatment protocol to prepare the gastrointestinal tract to receive the bacterial composition. In certain embodiments, the pretreatment protocol is advisable, such as when a patient has an acute infection with a highly resilient pathogen. In other embodiments, the pretreatment protocol is entirely optional, such as when the pathogen causing the infection is not resilient, or the patient has had an acute infection that has been successfully treated but where the physician is concerned that the infection may recur. In these instances, the pretreatment protocol can enhance the ability of the bacterial composition to affect the patient's microbiome.

As one way of preparing the patient for administration of the microbial ecosystem, at least one antibiotic can be administered to alter the bacteria in the patient. As another way of preparing the patient for administration of the microbial ecosystem, a standard colon-cleansing preparation can be administered to the patient to substantially empty the contents of the colon, such as used to prepare a patient for a colonscopy. By “substantially emptying the contents of the colon,” this application means removing at least 75%, at least 80%, at least 90%, at least 95%, or about 100% of the contents of the ordinary volume of colon contents. Antibiotic treatment can precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection, or if a patient has received an antibiotic as part of a specific pretreatment protocol, in one embodiment, the antibiotic can be stopped in sufficient time to allow the antibiotic to be substantially reduced in concentration in the gut before the bacterial composition is administered. In one embodiment, the antibiotic can be discontinued 1, 2, or 3 days before the administration of the bacterial composition. In another embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the bacterial composition. In another embodiment, the antibiotic can be chosen so the constituents in the bacterial composition have an MIC50 that is higher than the concentration of the antibiotic in the gut.

MIC50 of a bacterial composition or the elements in the composition can be determined by methods well known in the art. Reller et al., Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices, Clinical Infectious Diseases 49(11):1749-1755 (2009). In such an embodiment, the additional time between antibiotic administration and administration of the bacterial composition is not necessary. If the pretreatment protocol is part of treatment of an acute infection, the antibiotic can be chosen so that the infection is sensitive to the antibiotic, but the constituents in the bacterial composition are not sensitive to the antibiotic.

Routes of Administration

The bacterial compositions of the invention are suitable for administration to mammals and non-mammalian animals in need thereof. In certain embodiments, the mammalian subject is a human subject who has one or more symptoms of a dysbiosis.

When a mammalian subject is suffering from a disease, disorder or condition characterized by an aberrant microbiota, the bacterial compositions described herein are suitable for treatment thereof. In some embodiments, the mammalian subject has not received antibiotics in advance of treatment with the bacterial compositions. For example, the mammalian subject has not been administered at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has received one or more treatments with one or more different antibiotic compounds and such treatment(s) resulted in no improvement or a worsening of symptoms.

In some embodiments, the gastrointestinal disease, disorder or condition is diarrhea caused by C. difficile including recurrent C. difficile infection, ulcerative colitis, colitis, Crohn's disease, or irritable bowel disease. Beneficially, the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition. In some embodiments, the therapeutic composition is administered at intervals greater than two days, such as once every three, four, five or six days, or every week or less frequently than every week. In other embodiments, the preparation can be administered intermittently according to a set schedule, e.g., once a day, once weekly, or once monthly, or when the subject relapses from the primary illness. In another embodiment, the preparation may be administered on a long-term basis to subjects who are at risk for infection with or who may be carriers of these pathogens, including subjects who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care professionals).

In certain embodiments, the bacterial composition is administered enterically. This preferentially includes oral administration, or by an oral or nasal tube (including nasogastric, nasojejunal, oral gastric, or oral jejunal). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy). The bacterial composition can be administered to at least one region of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum. In some embodiments, it is administered to all regions of the gastrointestinal tract. The bacterial compositions can be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids. The bacterial compositions can also be administered in gel or liquid form by the oral route or through a nasogastric tube, or by the rectal route in a gel or liquid form, by enema or instillation through a colonoscope or by a suppository.

If the composition is administered colonoscopically and, optionally, if the bacterial composition is administered by other rectal routes (such as an enema or suppository) or even if the subject has an oral administration, the subject can have a colon-cleansing preparation. The colon-cleansing preparation can facilitate proper use of the colonoscope or other administration devices, but even when it does not serve a mechanical purpose, it can also maximize the proportion of the bacterial composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. Any ordinarily acceptable colon-cleansing preparation may be used such as those typically provided when a subject undergoes a colonoscopy.

Dosages and Schedule for Administration

In some embodiments, the bacteria and bacterial compositions are provided in a dosage form. In certain embodiments, the dosage form is designed for administration of at least one OTU or combination thereof disclosed herein, wherein the total amount of bacterial composition administered is selected from 0.1 ng to 10 g, 10 ng to 1 g, 100 ng to 0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg, or from 10-15 mg. In other embodiments, the bacterial composition is consumed at a rate of from 0.1 ng to 10 g a day, 10 ng to 1 g a day, 100 ng to 0.1 g a day, 0.1 mg to 500 mg a day, 1 mg to 100 mg a day, or from 10-15 mg a day, or more.

In certain embodiments, the treatment period is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. In some embodiments the treatment period is from 1 day to 1 week, from 1 week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months, from 6 months to 1 year, or for over a year.

In one embodiment, 105 and 1012 microorganisms total can be administered to the patient in a given dosage form. In another embodiment, an effective amount can be provided in from 1 to 500 ml or from 1 to 500 grams of the bacterial composition having from 107 to 1011 bacteria per ml or per gram, or a capsule, tablet or suppository having from 1 mg to 1000 mg lyophilized powder having from 107 to 1011 bacteria. Those receiving acute treatment can receive higher doses than those who are receiving chronic administration (such as hospital workers or those admitted into long-term care facilities).

Any of the preparations described herein can be administered once on a single occasion or on multiple occasions, such as once a day for several days or more than once a day on the day of administration (including twice daily, three times daily, or up to five times daily). In another embodiment, the preparation can be administered intermittently according to a set schedule, e.g., once weekly, once monthly, or when the patient relapses from the primary illness. In one embodiment, the preparation can be administered on a long-term basis to individuals who are at risk for infection with or who may be carriers of these pathogens, including individuals who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care professionals).

Patient Selection

Particular bacterial compositions can be selected for individual patients or for patients with particular profiles. For example, 16S sequencing can be performed for a given patient to identify the bacteria present in his or her microbiota. The sequencing can either profile the patient's entire microbiome using 16S sequencing (to the family, genera, or species level), a portion of the patient's microbiome using 16S sequencing, or it can be used to detect the presence or absence of specific candidate bacteria that are biomarkers for health or a particular disease state, such as markers of multi-drug resistant organisms or specific genera of concern such as Escherichia. Based on the biomarker data, a particular composition can be selected for administration to a patient to supplement or complement a patient's microbiota in order to restore health or treat or prevent disease. In another embodiment, patients can be screened to determine the composition of their microbiota to determine the likelihood of successful treatment.

Combination Therapy

The bacterial compositions can be administered with other agents in a combination therapy mode, including anti-microbial agents and prebiotics. Administration can be sequential, over a period of hours or days, or simultaneous.

In one embodiment, the bacterial compositions are included in combination therapy with one or more anti-microbial agents, which include anti-bacterial agents, anti-fungal agents, anti-viral agents and anti-parasitic agents.

Anti-bacterial agents can include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).

Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine, Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine, Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine, Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir, Zalcitabine, Zanamivir and Zidovudine.

Examples of antifungal compounds include, but are not limited to polyene antifungals such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin; imidazole antifungals such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; triazole antifungals such as fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and albaconazole; thiazole antifungals such as abafungin; allylamine antifungals such as terbinafine, naftifine, and butenafine; and echinocandin antifungals such as anidulafungin, caspofungin, and micafungin. Other compounds that have antifungal properties include, but are not limited to polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.

In one embodiment, the bacterial compositions are included in combination therapy with one or more corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines, and combinations thereof.

A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health. Prebiotics can include complex carbohydrates, amino acids, peptides, or other essential nutritional components for the survival of the bacterial composition. Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, inulin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide, and xylooligosaccharides.

Methods for Testing Bacterial Compositions for Populating Effect

In Vivo Assay for Determining Whether a Bacterial Composition Populates a Subject's Gastrointestinal Tract

In order to determine that the bacterial composition populates the gastrointestinal tract of a subject, an animal model, such as a mouse model, can be used. The model can begin by evaluating the microbiota of the mice. Qualitative assessments can be accomplished using 16S profiling of the microbial community in the feces of normal mice. It can also be accomplished by full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques. Quantitative assessments can be conducted using quantitative PCR (qPCR), described below, or by using traditional microbiological techniques and counting colony formation.

Optionally, the mice can receive an antibiotic treatment to mimic the condition of dysbiosis. Antibiotic treatment can decrease the taxonomic richness, diversity, and evenness of the community, including a reduction of abundance of a significant number of bacterial taxa. Dethlefsen et al., The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology 6(11):3280 (2008). At least one antibiotic can be used, and antibiotics are well known. Antibiotics can include aminoglycoside antibiotic (amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), amoxicillin, ampicillin, Augmentin (an amoxicillin/clavulanate potassium combination), cephalosporin (cefaclor, defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime, cefuroxime, cephalexin), clavulanate potassium, clindamycin, colistin, gentamycin, kanamycin, metronidazole, or vancomycin. As an individual, nonlimiting specific example, the mice can be provided with drinking water containing a mixture of the antibiotics kanamycin, colistin, gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5 mg/kg, 21.5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight), respectively, for 7 days. Alternatively, mice can be administered ciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse body weight), for 7 days. If the mice are provided with an antibiotic, a wash out period of from one day to three days may be provided with no antibiotic treatment and no bacterial composition treatment.

Subsequently, the test bacterial composition is administered to the mice by oral gavage. The test bacterial composition may be administered in a volume of 0.2 ml containing 10⁴ CFUs of each type of bacteria in the bacterial composition. Dose-response may be assessed by using a range of doses, including, but not limited to 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, and/or 10¹⁰.

The mice can be evaluated using 16S sequencing, full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. For example only, one day, three days, one week, two weeks, and one month after administration of the bacterial composition to the mice, 16S profiling is conducted to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. Quantitative assessments, including qPCR and traditional microbiological techniques such as colony counting, can additionally or alternatively be performed, at the same time intervals.

Furthermore, the number of sequence counts that correspond exactly to those in the bacterial composition over time can be assessed to determine specifically which components of the bacterial composition reside in the gastrointestinal tract over a particular period of time. In one embodiment, the strains of the bacterial composition persist for a desired period of time. In another embodiment, the components of the bacterial composition persist for a desired period of time, while also increasing the ability of other microbes (such as those present in the environment, food, etc.) to populate the gastrointestinal tract, further increasing overall diversity, as discussed below.

Ability of Bacterial Compositions to Populate Different Regions of the Gastrointestinal Tract

The present bacterial compositions can also be assessed for their ability to populate different regions on the gastrointestinal tract. In one embodiment, a bacterial composition can be chosen for its ability to populate one or more than one region of the gastrointestinal tract, including, but not limited to the stomach, the small intestine (duodenum, jejunum, and ileum), the large intestine (the cecum, the colon (the ascending, transverse, descending, and sigmoid colon), and the rectum).

An in vivo study can be conducted to determine which regions of the gastrointestinal tract a given bacterial composition will populate. A mouse model similar to the one described above can be conducted, except instead of assessing the feces produced by the mice, particular regions of the gastrointestinal tract can be removed and studied individually. For example, at least one particular region of the gastrointestinal tract can be removed and a qualitative or quantitative determination can be performed on the contents of that region of the gastrointestinal tract. In another embodiment, the contents can optionally be removed and the qualitative or quantitative determination may be conducted on the tissue removed from the mouse.

qPCR

As one quantitative method for determining whether a bacterial composition populates the gastrointestinal tract, quantitative PCR (qPCR) can be performed. Standard techniques can be followed to generate a standard curve for the bacterial composition of interest, either for all of the components of the bacterial composition collectively, individually, or in subsets (if applicable). Genomic DNA can be extracted from samples using commercially-available kits, such as the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME, Gaithersburg, Md.) and primers specific for the bacterial composition of interest, and may be conducted on a MicroAmp® Fast Optical 96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island, N.Y.) and performed on a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System (BioRad, Hercules, Calif.), with fluorescent readings of the FAM and ROX channels. The Cq value for each well on the FAM channel is determined by the CFX Manager™ software version 2.1. The log₁₀(cfu/ml) of each experimental sample is calculated by inputting a given sample's Cq value into linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log₁₀(cfu/ml) of those samples. The skilled artisan may employ alternative qPCR modes.

Methods for Characterization of Bacterial Compositions

In certain embodiments, provided are methods for testing certain characteristics of bacterial compositions. For example, the sensitivity of bacterial compositions to certain environmental variables is determined, e.g., in order to select for particular desirable characteristics in a given composition, formulation and/or use. For example, the constituents in the bacterial composition can be tested for pH resistance, bile acid resistance, and/or antibiotic sensitivity, either individually on a constituent-by-constituent basis or collectively as a bacterial composition comprised of multiple bacterial constituents (collectively referred to in this section as bacterial composition).

pH Sensitivity Testing

If a bacterial composition will be administered other than to the colon or rectum (i.e., for example, an oral route), optionally testing for pH resistance enhances the selection of bacterial compositions that will survive at the highest yield possible through the varying pH environments of the distinct regions of the GI tract. Understanding how the bacterial compositions react to the pH of the GI tract also assists in formulation, so that the number of bacteria in a dosage form can be increased if beneficial and/or so that the composition may be administered in an enteric-coated capsule or tablet or with a buffering or protective composition. As the pH of the stomach can drop to a pH of 1 to 2 after a high-protein meal for a short time before physiological mechanisms adjust it to a pH of 3 to 4 and often resides at a resting pH of 4 to 5, and as the pH of the small intestine can range from a pH of 6 to 7.4, bacterial compositions can be prepared that survive these varying pH ranges (specifically wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of the bacteria can survive gut transit times through various pH ranges). This can be tested by exposing the bacterial composition to varying pH ranges for the expected gut transit times through those pH ranges. Therefore, as a nonlimiting example only, 18-hour cultures of bacterial compositions can be grown in standard media, such as gut microbiota medium (“GMM”, see Goodman et al., Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or another animal-products-free medium, with the addition of pH adjusting agents for a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4 to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. An alternative method for testing stability to acid is described in U.S. Pat. No. 4,839,281. Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.

Bile Acid Sensitivity Testing

Additionally, in some embodiments, testing for bile-acid resistance enhances the selection of bacterial compositions that will survive exposures to bile acid during transit through the GI tract. Bile acids are secreted into the small intestine and can, like pH, affect the survival of bacterial compositions. This can be tested by exposing the bacterial compositions to bile acids for the expected gut exposure time to bile acids. For example, bile acid solutions can be prepared at desired concentrations using 0.05 mM Tris at pH 9 as the solvent. After the bile acid is dissolved, the pH of the solution may be adjusted to 7.2 with 10% HCl. Bacterial compositions can be cultured in 2.2 ml of a bile acid composition mimicking the concentration and type of bile acids in the patient, 1.0 ml of 10% sterile-filtered feces media and 0.1 ml of an 18-hour culture of the given strain of bacteria. Incubations may be conducted for from 2.5 to 3 hours or longer. An alternative method for testing stability to bile acid is described in U.S. Pat. No. 4,839,281. Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.

Antibiotic Sensitivity Testing

As a further optional sensitivity test, bacterial compositions can be tested for sensitivity to antibiotics. In one embodiment, bacterial compositions can be chosen so that the bacterial constituents are sensitive to antibiotics such that if necessary they can be eliminated or substantially reduced from the patient's gastrointestinal tract by at least one antibiotic targeting the bacterial composition.

Adherence to Gastrointestinal Cells

The bacterial compositions may optionally be tested for the ability to adhere to gastrointestinal cells. A method for testing adherence to gastrointestinal cells is described in U.S. Pat. No. 4,839,281.

Methods for Purifying Spores

Solvent Treatments

To purify the bacterial spores, the fecal material is subjected to one or more solvent treatments. A solvent treatment is a miscible solvent treatment (either partially miscible or fully miscible) or an immiscible solvent treatment. Miscibility is the ability of two liquids to mix with each to form a homogeneous solution. Water and ethanol, for example, are fully miscible such that a mixture containing water and ethanol in any ratio will show only one phase. Miscibility is provided as a wt/wt %, or weight of one solvent in 100 g of final solution. If two solvents are fully miscible in all proportions, their miscibility is 100%. Provided as fully miscible solutions with water are alcohols, e.g., methanol, ethanol, isopropanol, butanol, etc. The alcohols can be provided already combined with water; e.g., a solution containing 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% or greater than 95% Other solvents are only partially miscible, meaning that only some portion will dissolve in water. Diethyl ether, for example, is partially miscible with water. Up to 7 grams of diethyl ether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. If more diethyl ether is added, a two phase solution will result with a distinct diethyl ether layer above the water. Other miscible materials include ethers, dimethoxyethane, or tetrahydrofuran In contrast, an oil such as an alkane and water are immiscible and form two phases. Further, immiscible treatments are optionally combined with a detergent, either an ionic detergent or a non-ionic detergent. Exemplary detergents include Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or a polyol.

Chromatography Treatments

To purify spore populations, the fecal materials are subjected to one or more chromatographic treatments, either sequentially or in parallel. In a chromatographic treatment, a solution containing the fecal material is contacted with a solid medium containing a hydrophobic interaction chromatographic (HIC) medium or an affinity chromatographic medium. In an alternative embodiment, a solid medium capable of absorbing a residual habitat product present in the fecal material is contacted with a solid medium that adsorbs a residual habitat product. In certain embodiments, the HIC medium contains sepharose or a derivatized sepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, or butyl-s sepharose. In other embodiments, the affinity chromatographic medium contains material derivatized with mucin type I, II, III, IV, V, or VI, or oligosaccharides derived from or similar to those of mucins type I, II, III, IV, V, or VI. Alternatively, the affinity chromatographic medium contains material derivatized with antibodies that recognize spore-forming bacteria.

Mechanical Treatments

Provided herein is the physical disruption of the fecal material, particularly by one or more mechanical treatment such as blending, mixing, shaking, vortexing, impact pulverization, and sonication. As provided herein, the mechanical disrupting treatment substantially disrupts a non-spore material present in the fecal material and does not substantially disrupt a spore present in the fecal material. Mechanical treatments optionally include filtration treatments, where the desired spore populations are retained on a filter while the undesirable (non-spore) fecal components to pass through, and the spore fraction is then recovered from the filter medium. Alternatively, undesirable particulates and eukaryotic cells may be retained on a filter while bacterial cells including spores pass through. In some embodiments the spore fraction retained on the filter medium is subjected to a diafiltration step, wherein the retained spores are contacted with a wash liquid, typically a sterile saline-containing solution or other diluent, in order to further reduce or remove the undesirable fecal components.

Thermal Treatments

Provided herein is the thermal disruption of the fecal material. Generally, the fecal material is mixed in a saline-containing solution such as phosphate-buffered saline (PBS) and subjected to a heated environment, such as a warm room, incubator, water-bath, or the like, such that efficient heat transfer occurs between the heated environment and the fecal material. Preferably the fecal material solution is mixed during the incubation to enhance thermal conductivity and disrupt particulate aggregates. Thermal treatments can be modulated by the temperature of the environment and/or the duration of the thermal treatment. For example, the fecal material or a liquid comprising the fecal material is subjected to a heated environment, e.g., a hot water bath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at least about 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours. In certain embodiments the thermal treatment occurs at two different temperatures, such as 30 seconds in a 100 degree Celsius environment followed by 10 minutes in a 50 degree Celsius environment. In preferred embodiments the temperature and duration of the thermal treatment are sufficient to kill or remove pathogenic materials while not substantially damaging or reducing the germination-competency of the spores.

Irradiation Treatments

Provided are methods of treating the fecal material or separated contents of the fecal material with ionizing radiation, typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations. For example, ultraviolet radiation at 254 nm provided at an energy level below about 22,000 microwatt seconds per cm² will not generally destroy desired spores.

Centrifugation and Density Separation Treatments

Provided are methods of separating desired spore populations from the other components of the fecal material by centrifugation. A solution containing the fecal material is subjected to one or more centrifugation treatments, e.g., at about 1000×g, 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g. Differential centrifugation separates desired spores from undesired non-spore material; at low forces the spores are retained in solution, while at higher forces the spores are pelleted while smaller impurities (e.g., virus particles, phage) are retained in solution. For example, a first low force centrifugation pellets fibrous materials; a second, higher force centrifugation pellets undesired eukaryotic cells, and a third, still higher force centrifugation pellets the desired spores while small contaminants remain in suspension. In some embodiments density or mobility gradients or cushions (e.g., step cushions), such as Percoll, Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separate desired spore populations from other materials in the fecal material.

Also provided herein are methods of producing spore populations that combine two or more of the treatments described herein in order to synergistically purify the desired spores while killing or removing undesired materials and/or activities from the spore population. It is generally desirable to retain the spore populations under non-germinating and non-growth promoting conditions and media, in order to minimize the growth of pathogenic bacteria present in the spore populations and to minimize the germination of spores into vegetative bacterial cells.

PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS OF THE INVENTION

Formulations

Provided are formulations for administration to humans and other subjects in need thereof. Generally the bacterial compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.

In some embodiments, the composition comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula C_(n)H_(2n)O_(n). A carbohydrate can be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates can contain modified saccharide units, such as 2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group is replace with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydrates can exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. As used herein, a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments, the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In other embodiments, the composition comprises at least one modified lipid, for example, a lipid that has been modified by cooking.

In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

In certain embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.

In other embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.

In another embodiment, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.

In other embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.

In another embodiment, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

In other embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as an excipient. In other embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. In another embodiment, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

In another embodiment, the excipient comprises a flavoring agent. Flavoring agents can be chosen from synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof. In some embodiments the flavoring agent is selected from cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

In other embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as the sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like. Also contemplated are hydrogenated starch hydrolysates and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly the potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In yet other embodiments, the composition comprises a coloring agent. Non-limiting examples of suitable color agents include food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C). The coloring agents can be used as dyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in the formulation is usually about 99% or less, such as about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less of the total weight of the composition.

The bacterial compositions disclosed herein can be formulated into a variety of forms and administered by a number of different means. The compositions can be administered orally, rectally, or parenterally, in formulations containing conventionally acceptable carriers, adjuvants, and vehicles as desired. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques. In an exemplary embodiment, the bacterial composition is administered orally.

Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. A capsule typically comprises a core material comprising a bacterial composition and a shell wall that encapsulates the core material. In some embodiments, the core material comprises at least one of a solid, a liquid, and an emulsion. In other embodiments, the shell wall material comprises at least one of a soft gelatin, a hard gelatin, and a polymer. Suitable polymers include, but are not limited to: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g., those copolymers sold under the trade name “Eudragit”); vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac). In yet other embodiments, at least one polymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed, multiply layered, and/or coated. The coating can be single or multiple. In one embodiment, the coating material comprises at least one of a saccharide, a polysaccharide, and glycoproteins extracted from at least one of a plant, a fungus, and a microbe. Non-limiting examples include corn starch, wheat starch, potato starch, tapioca starch, cellulose, hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin, mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gum karaya, gum ghatti, tragacanth gum, funori, carrageenans, agar, alginates, chitosans, or gellan gum. In some embodiments the coating material comprises a protein. In another embodiment, the coating material comprises at least one of a fat and an oil. In other embodiments, the at least one of a fat and an oil is high temperature melting. In yet another embodiment, the at least one of a fat and an oil is hydrogenated or partially hydrogenated. In one embodiment, the at least one of a fat and an oil is derived from a plant. In other embodiments, the at least one of a fat and an oil comprises at least one of glycerides, free fatty acids, and fatty acid esters. In some embodiments, the coating material comprises at least one edible wax. The edible wax can be derived from animals, insects, or plants. Non-limiting examples include beeswax, lanolin, bayberry wax, carnauba wax, and rice bran wax. Tablets and pills can additionally be prepared with enteric coatings.

Alternatively, powders or granules embodying the bacterial compositions disclosed herein can be incorporated into a food product. In some embodiments, the food product is a drink for oral administration. Non-limiting examples of a suitable drink include fruit juice, a fruit drink, an artificially flavored drink, an artificially sweetened drink, a carbonated beverage, a sports drink, a liquid diary product, a shake, an alcoholic beverage, a caffeinated beverage, infant formula and so forth. Other suitable means for oral administration include aqueous and nonaqueous solutions, emulsions, suspensions and solutions and/or suspensions reconstituted from non-effervescent granules, containing at least one of suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, coloring agents, and flavoring agents.

In some embodiments, the food product can be a solid foodstuff. Suitable examples of a solid foodstuff include without limitation a food bar, a snack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, a frozen yogurt bar, and the like.

In other embodiments, the compositions disclosed herein are incorporated into a therapeutic food. In some embodiments, the therapeutic food is a ready-to-use food that optionally contains some or all essential macronutrients and micronutrients. In another embodiment, the compositions disclosed herein are incorporated into a supplementary food that is designed to be blended into an existing meal. In one embodiment, the supplemental food contains some or all essential macronutrients and micronutrients. In another embodiment, the bacterial compositions disclosed herein are blended with or added to an existing food to fortify the food's protein nutrition. Examples include food staples (grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea, soda, beer, liquor, sports drinks), snacks, sweets and other foods.

In one embodiment, the formulations are filled into gelatin capsules for oral administration. An example of an appropriate capsule is a 250 mg gelatin capsule containing from 10 (up to 100 mg) of lyophilized powder (10⁸ to 10¹¹ bacteria), 160 mg microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate. In an alternative embodiment, from 10⁵ to 10¹² bacteria may be used, 10⁵ to 10⁷, 10⁶ to 10⁷, or 10⁸ to 10¹⁰, with attendant adjustments of the excipients if necessary. In an alternative embodiment, an enteric-coated capsule or tablet or with a buffering or protective composition can be used.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed. (Plenum Press) Vols A and B(1992).

Example 1. Provision of Fecal Material

Fresh fecal samples were obtained from healthy human donors who have been screened for general good health and for the absence of infectious diseases, and meet inclusion and exclusion criteria, inclusion criteria include being in good general health, without significant medical history, physical examination findings, or clinical laboratory abnormalities, regular bowel movements with stool appearance typically Type 2, 3, 4, 5 or 6 on the Bristol Stool Scale, and having a BMI ≥18 kg/m² and ≤25 kg/m². Exclusion criteria generally included significant chronic or acute medical conditions including renal, hepatic, pulmonary, gastrointestinal, cardiovascular, genitourinary, endocrine, immunologic, metabolic, neurologic or hematological disease, a family history of, inflammatory bowel disease including Crohn's disease and ulcerative colitis, Irritable bowel syndrome, colon, stomach or other gastrointestinal malignancies, or gastrointestinal polyposis syndromes, or recent use of yogurt or commercial probiotic materials in which an organism(s) is a primary component. Samples were collected directly using a commode specimen collection system, which contains a plastic support placed on the toilet seat and a collection container that rests on the support. Feces were deposited into the container, and the lid was then placed on the container and sealed tightly. The sample was then delivered on ice within 1-4 hours for processing. Samples were mixed with a sterile disposable tool, and 2-4 g aliquots were weighed and placed into tubes and flash frozen in a dry ice/ethanol bath. Aliquots are frozen at −80 degrees Celsius until use.

Optionally, the fecal material was suspended in a solution, and/or fibrous and/or particulate materials were removed. A frozen aliquot containing a known weight of feces was removed from storage at −80 degrees Celsius and allowed to thaw at room temperature. Sterile 1×PBS was added to create a 10% w/v suspension, and vigorous vortexing was performed to suspend the fecal material until the material appeared homogeneous. The material was then left to sit for 10 minutes at room temperature to sediment fibrous and particulate matter. The suspension above the sediment was then carefully removed into a new tube and contains a purified spore population. Optionally, the suspension was then centrifuged at a low speed, e.g., 1000×g, for 5 minutes to pellet particulate matter including fibers. The pellet was discarded and the supernatant, which contained vegetative organisms and spores, was removed into a new tube. The supernatant was then centrifuged at 6000×g for 10 minutes to pellet the vegetative organisms and spores. The pellet was then resuspended in 1×PBS with vigorous vortexing until the material appears homogenous.

Example 2. Spore Purification from Alcohol Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated at 37 degrees Celsius for 1 hour. After incubation the suspension was centrifuged at 13,000 rpm for 5 minutes to pellet spores. The supernatant was discarded and the pellet was resuspended in an equal volume of PBS. Glycerol was added to a final concentration of 15% and then the purified spore fraction is stored at −80 degrees Celsius.

Example 2A. Generation of a Spore Preparation from Alcohol Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated at 37 degrees Celsius for 1 hour. After incubation the suspension is centrifuged at 13,000 rpm for 5 minutes to concentrate spores into a pellet containing a purified sprore-containing preparation. The supernatant was discarded and the pellet resuspended in an equal volume of PBS. Glycerol was added to a final concentration of 15% and then the purified spore preparation was stored at −80 degrees Celsius.

Example 3. Spore Purification from Thermal Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS was incubated in a water bath at 80 degrees Celsius for 30 minutes. Glycerol was added to a final concentration of 15% and then the enriched spore containing material was stored at −80 degrees Celsius.

Example 4. Spore Purification from Alcohol Treatment and Thermal Treatment of Fecal Material

A 10% w/v suspension of human feces in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated in a water bath under aerobic conditions at 37 degrees Celsius for 1 hour. After incubation the suspension was centrifuged at 13,000 rpm for 5 minutes to pellet spores. The supernatant was discarded and the pellet was resuspended in equal volume PBS. The ethanol treated spore population was then incubated in a water bath at 80 degrees Celsius for 30 minutes. Glycerol was added to a final concentration of 15% and the purified spore fraction was stored at −80 C.

Example 5. Spore Purification from Detergent Treatment of Fecal Material

A 10% w/v suspension of human feces in PBS is prepared to contain a final concentration of 0.5 to 2% Triton X-100. After shaking incubation for 30 minutes at 25 to 37 degrees Celsius, the sample is centrifuged at 1000 g for 5-10 minutes to pellet particulate matter and large cells. The bacterial spores are recovered in the supernatant fraction, where the purified spore population is optionally further treated, such as in Example 4. Without being bound by theory, detergent addition to the fecal mixture produces better spore populations, at least in part by enhancing separation of the spores from particulates thereby resulting in higher yields of spores.

Example 6. Spore Purification by Chromatographic Separation of Fecal Material

A spore-enriched population such as obtained from Examples 1-5 above, is mixed with NaCl to a final concentration of 4M total salt and contacted with octyl Sepharose 4 Fast Flow to bind the hydrophobic spore fraction. The resin is washed with 4M NaCl to remove less hydrophobic components, and the spores are eluted with distilled water, and the desired enriched spore fraction is collected via UV absorbance.

Example 7. Spore Purification by Filtration of Fecal Material

A spore-enriched population such as obtained from Examples 1-6 above is diluted 1:10 with PBS, and placed in the reservoir vessel of a tangential flow microfiltration system. A 0.2 um pore size mixed cellulose ester hydrophilic tangential flow filter is connected to the reservoir such as by a tubing loop. The diluted spore preparation is recirculated through the loop by pumping, and the pressure gradient across the walls of the microfilter forces the supernatant liquid through the filter pores. By appropriate selection of the filter pore size the desired bacterial spores are retained, while smaller contaminants such as cellular debris, and other contaminants in feces such as bacteriophage pass through the filter. Fresh PBS buffer is added to the reservoir periodically to enhance the washout of the contaminants. At the end of the diafiltration, the spores are concentrated approximately ten-fold to the original concentration. The purified spores are collected from the reservoir and stored as provided herein.

Example 8. Characterization of Purified Spore Populations

Counts of viable spores are determined by performing 10 fold serial dilutions in PBS and plating to Brucella Blood Agar Petri plates or applicable solid media. Plates are incubated at 37 degrees Celsius for 2 days. Colonies are counted from a dilution plate with 50-400 colonies and used to back-calculate the number of viable spores in the population. The ability to germinate into vegetative bacteria is also demonstrated. Visual counts are determined by phase contrast microscopy. A spore preparation is either diluted in PBS or concentrated by centrifugation, and a 5 microliter aliquot is placed into a Petroff Hauser counting chamber for visualization at 400× magnification. Spores are counted within ten 0.05 mm×0.05 mm grids and an average spore count per grid is determined and used to calculate a spore count per ml of preparation. Lipopolysaccharide (LPS) reduction in purified spore populations is measured using a Limulus amebocyte lysate (LAL) assay such as the commercially available ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, N.J.) or other standard methods known to those skilled in the art.

Example 9. Determination of Bacterial Pathogens in Purified Spore Populations

Bacterial pathogens present in a purified spore population are determined by qPCR using specific oligonucleotide primers as follows.

Standard Curve Preparation. The standard curve is generated from wells containing the pathogen of interest at a known concentration or simultaneously quantified by selective spot plating. Serial dilutions of duplicate cultures are performed in sterile phosphate-buffered saline. Genomic DNA is then extracted from the standard curve samples along with the other samples.

Genomic DNA Extraction.

Genomic DNA may be extracted from 100 μl of fecal samples, fecal-derived samples, or purified spore preparations using the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) according to the manufacturer's instructions with two exceptions: the beadbeating is performed for 2×4:40 minutes using a BioSpec Mini-Beadbeater-96 (BioSpec Products, Bartlesville, Okla.) and the DNA is eluted in 50 μl of Solution C6. Alternatively the genomic DNA could be isolated using the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), the Sigma-Aldrich Extract-N-Amp™ Plant PCR Kit, the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

[qPCR Composition and Conditions.

The qPCR reaction to detect C. difficile contains 1× HotMasterMix (SPRIME, Gaithersburg, Md.), 900 nM of Wr-tcdB-F (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG, (SEQ ID NO: 2040) IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R (CATGCTTTTTTAGTTTCTGGATTGAA, (SEQ ID NO: 2041) IDT, Coralville, Iowa), 250 nM of We-tcdB-P (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 2042), Life Technologies, Grand Island, N.Y.), and PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl (Primers adaped from: Wroblewski, D. et al. Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens. Journal of Clinical Microbiology 47:2142-2148 (2009)). This reaction mixture is aliquoted to wells of a MicroAmp® Fast Optical 96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island, N.Y.). To this reaction mixture, 2 μl of extracted genomic DNA is added. The qPCR is performed on a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System (BioRad, Hercules, Calif.). The thermocycling conditions are 95° C. for 2 minutes followed by 45 cycles of 95° C. for 3 seconds, 60° C. for 30 seconds, and fluorescent readings of the FAM and ROX channels. Other bacterial pathogens can be detected by using primers and a probe specific for the pathogen of interest.

Data Analysis.

The Cq value for each well on the FAM channel is determined by the CFX Manager™ Software Version 2.1. The log 10(cfu/ml) of each experimental sample is calculated by inputting a given sample's Cq value into linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log 10(cfu/ml) of those samples.

[Viral pathogens present in a purified spore population are determined by qPCR as described herein and otherwise known in the art.

Example 10: Species Identification

The identity of the spore-forming species which grew up from a complex fraction can be determined in multiple ways. First, individual colonies can be picked into liquid media in a 96 well format, grown up and saved as 15% glycerol stocks at −80 C. Aliquots of the cultures can be placed into cell lysis buffer and colony PCR methods can be used to amplify and sequence the 16S rDNA gene (Example 2). Alternatively, colonies may be streaked to purity in several passages on solid media. Well separated colonies are streaked onto the fresh plates of the same kind and incubated for 48-72 hours at 37 C. The process is repeated multiple times in order to ensure purity. Pure cultures can be analyzed by phenotypic- or sequence-based methods, including 16S rDNA amplification and sequencing as described in Examples 11 & 12. Sequence characterization of pure isolates or mixed communities e.g. plate scrapes and spore fractions can also include whole genome shotgun sequencing. The latter is valuable to determine the presence of genes associated with sporulation, antibiotic resistance, pathogenicity, and virulence. Colonies can also be scraped from plates en masse and sequenced using a massively parallel sequencing method as described in Examples 11 & 12 such that individual 16S signatures can be identified in a complex mixture. Optionally, the sample can be sequenced prior to germination (if appropriate DNA isolation procedures are used to lsye and release the DNA from spores) in order to compare the diversity of germinable species with the total number of species in a spore sample. As an alternative or complementary approach to 16S analysis, MALDI-TOF-mass spec can also be used for species identification (as reviewed in Anaerobe 22:123).

Example 11: 16s Sequencing to Determine Operational Taxonomic Unit (OTU)

Method for Determining 16S Sequence

OTUs may be defined either by full 16S sequencing of the rRNA gene, by sequencing of a specific hypervariable region of this gene (i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination of hypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial 16S rDNA is approximately 1500 nucleotides in length and is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most microbes.

Using well known techniques, in order to determine the full 16S sequence or the sequence of any hypervariable region of the 16S sequence, genomic DNA is extracted from a bacterial sample, the 16S rDNA (full region or specific hypervariable regions) amplified using polymerase chain reaction (PCR), the PCR products cleaned, and nucleotide sequences delineated to determine the genetic composition of 16S gene or subdomain of the gene. If full 16S sequencing is performed, the sequencing method used may be, but is not limited to, Sanger sequencing. If one or more hypervariable regions are used, such as the V4 region, the sequencing may be, but is not limited to being, performed using the Sanger method or using a next-generation sequencing method, such as an IIlumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.

In addition to the 16S rRNA gene, one may define an OTU by sequencing a selected set of genes that are known to be marker genes for a given species or taxonomic group of OTUs. These genes may alternatively be assayed using a PCR-based screening strategy. As example, various strains of pathogenic Escherichia coli can be distinguished using DNAs from the genes that encode heat-labile (LTI, LTIIa, and LTIIb) and heat-stable (STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attaching and effacing mechanisms (eaeA), enteroaggregative mechanisms (Eagg), and enteroinvasive mechanisms (Einv). The optimal genes to utilize for taxonomic assignment of OTUs by use of marker genes will be familiar to one with ordinary skill of the art of sequence based taxonomic identification.

Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hot alkaline lysis method. 1 μl of microbial culture is added to 9 μl of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at 95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. and neutralized by the addition of 10 μl of Neutralization Buffer (40 mM Tris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl). Alternatively, genomic DNA is extracted from pure microbial cultures using commercially available kits such as the Mo Bio Ultraclean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) or by standard methods known to those skilled in the art.

Amplification of 16S Sequences for Downstream Sanger Sequencing

To amplify bacterial 16S rDNA (FIG. 1A), 2 μl of extracted gDNA is added to a 20 μl final volume PCR reaction. For full-length 16 sequencing the PCR reaction also contains 1× HotMasterMix (5PRIME, Gaithersburg, Md.), 250 nM of 27f (AGRGTTTGATCMTGGCTCAG (SEQ ID NO: 2033), IDT, Coralville, Iowa), and 250 nM of 1492r (TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 2034), IDT, Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the volume. Alternatively, other universal bacterial primers or thermostable polymerases known to those skilled in the art are used. For example primers are available to those skilled in the art for the sequencing of the the “V1-V9 regions” of the 16S rRNA (FIG. 1A). These regions refer to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature. Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU. A person of ordinary skill in the art can identify the specific hypervariable regions of a candidate 16S rRNA (in FIG. 1A) by comparing the candidate sequence in question to the reference sequence (FIG. 1B) and identifying the hypervariable regions based on similarity to the reference hypervariable regions.

The PCR is performed on commercially available thermocyclers such as a BioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). The reactions are run at 94° C. for 2 minutes followed by 30 cycles of 94° C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30 seconds, followed by a 7 minute extension at 72° C. and an indefinite hold at 4° C. Following PCR, gel electrophoresis of a portion of the reaction products is used to confirm successful amplification of a ˜1.5 kb product.

To remove nucleotides and oligonucleotides from the PCR products, 2 μl of HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 5 μl of PCR product followed by a 15 minute incubation at 37° C. and then a 15 minute inactivation at 80° C.

Amplification of 16S Sequences for Downstream Characterization by Massively Parallel Sequencing Technologies

Amplification performed for downstream sequencing by short read technologies such as Illumina require amplification using primers known to those skilled in the art that additionally include a sequence-based barcoded tag. As example, to amplify the 16s hypervariable region V4 region of bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20 μl final volume PCR reaction. The PCR reaction also contains 1× HotMasterMix (SPRIME, Gaithersburg, Md.), 200 nM of V4_515f_adapt (AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCC GCGGTAA (SEQ ID NO: 2035), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc (CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 2036)_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 2037), IDT, Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the volume. These primers incorporate barcoded adapters for Illumina sequencing by synthesis. Optionally, identical replicate, triplicate, or quadruplicate reactions may be performed. Alternatively other universal bacterial primers or thermostable polymerases known to those skilled in the art are used to obtain different amplification and sequencing error rates as well as results on alternative sequencing technologies.

The PCR amplification is performed on commercially available thermocyclers such as a BioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). The reactions are run at 94° C. for 3 minutes followed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute, and 72° C. for 1 minute 30 seconds, followed by a 10 minute extension at 72° C. and a indefinite hold at 4° C. Following PCR, gel electrophoresis of a portion of the reaction products is used to confirm successful amplification of a ˜1.5 kb product. PCR cleanup is performed as specified in the previous example.

Sanger Sequencing of Target Amplicons from Pure Homogeneous Samples

To detect nucleic acids for each sample, two sequencing reactions are performed to generate a forward and reverse sequencing read. For full-length 16s sequencing primers 27f and 1492r are used. 40 ng of ExoSap-IT-cleaned PCR products are mixed with 25 pmol of sequencing primer and Mo Bio Molecular Biology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 15 μl total volume. This reaction is submitted to a commercial sequencing organization such as Genewiz (South Plainfield, N.J.) for Sanger sequencing.

Massively Parallel Sequencing of Target Amplicons from Heterogeneous Samples

DNA Quantification & Library Construction. The cleaned PCR amplification products are quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island, N.Y.) according to the manufacturer's instructions. Following quantification, the barcoded cleaned PCR products are combined such that each distinct PCR product is at an equimolar ratio to create a prepared Illumina library.

Nucleic Acid Detection. The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers (Illumina, San Diego, Calif.) with cluster generation, template hybridization, iso-thermal amplification, linearization, blocking and denaturization and hybridization of the sequencing primers performed according to the manufacturer's instructions. 16SV4SeqFw (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 2038)), 16SV4SeqRev (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 2037)), and 16SV4Index (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 2039)) (IDT, Coralville, Iowa) are used for sequencing. Other sequencing technologies can be used such as but not limited to 454, Pacific Biosciences, Helicos, Ion Torrent, and Nanopore using protocols that are standard to someone skilled in the art of genomic sequencing.

Example 12: Sequence Read Annotation

Primary Read Annotation

Nucleic acid sequences are analyzed and annotations are to define taxonomic assignments using sequence similarity and phylogenetic placement methods or a combination of the two strategies. A similar approach can be used to annotate protein names, transcription factor names, and any other classification schema for nucleic acid sequences. Sequence similarity based methods include those familiar to individuals skilled in the art including, but not limited to BLAST, BLASTx, tBLASTn, tBLASTx, RDP-classifier, DNAclust, and various implementations of these algorithms such as Qiime or Mothur. These methods rely on mapping a sequence read to a reference database and selecting the match with the best score and e-value. Common databases include, but are not limited to the Human Microbiome Project, NCBI non-redundant database, Greengenes, RDP, and Silva. Phylogenetic methods can be used in combination with sequence similarity methods to improve the calling accuracy of an annotation or taxonomic assignment. Here tree topologies and nodal structure are used to refine the resolution of the analysis. In this approach we analyze nucleic acid sequences using one of numerous sequence similarity approaches and leverage phylogenetic methods that are well known to those skilled in the art, including but not limited to maximum likelihood phylogenetic reconstruction (see e.g. Liu K, Linder C R, and Warnow T. 2011. RAxML and FastTree: Comparing Two Methods for Large-Scale Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731. McGuire G, Denham M C, and Balding D J. 2001. Models of sequence evolution for DNA sequences containing gaps. Mol. Biol. Evol 18: 481-490. Wróbel B. 2008. Statistical measures of uncertainty for branches in phylogenetic trees inferred from molecular sequences by using model-based methods. J. Appl. Genet. 49: 49-67.) Sequence reads are placed into a reference phylogeny comprised of appropriate reference sequences. Annotations are made based on the placement of the read in the phylogenetic tree. The certainty or significance of the OTU annotation is defined based on the OTU's sequence similarity to a reference nucleic acid sequence and the proximity of the OTU sequence relative to one or more reference sequences in the phylogeny. As an example, the specificity of a taxonomic assignment is defined with confidence at the the level of Family, Genus, Species, or Strain with the confidence determined based on the position of bootstrap supported branches in the reference phylogenetic tree relative to the placement of the OTU sequence being interrogated.

Clade Assignments

The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolving power of the 16S-V4 region of the 16S gene for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation. Given the topological nature of a phylogenetic tree and the fact that tree represents hierarchical relationships of OTUs to one another based on their sequence similarity and an underlying evolutionary model, taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1). Using this approach, clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood or other phylogenetic models familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another (generally, 1-5 bootstraps), and (ii) within a 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. The power of clade based analysis is that members of the same clade, due to their evolutionary relatedness, are likely to play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention.

Notably, 16S sequences of isolates of a given OTU are phylogenetically placed within their respective clades, sometimes in conflict with the microbiological-based assignment of species and genus that may have preceded 16S-based assignment. Discrepancies between taxonomic assignment based on microbiological characteristics versus genetic sequencing are known to exist from the literature.

Example 13: Germinating Spores

Germinating a spore fraction increases the number of viable spores that will grow on various media types. To germinate a population of spores, the sample is moved to the anaerobic chamber, resuspended in prereduced PBS, mixed and incubated for 1 hour at 37 C to allow for germination. Germinants can include amino-acids (e.g., alanine, glycine), sugars (e.g., fructose), nucleosides (e.g., inosine), bile salts (e.g., cholate and taurocholate), metal cations (e.g., Mg2+, Ca2+), fatty acids, and long-chain alkyl amines (e.g., dodecylamine, Germination of bacterial spores with alkyl primary amines” J. Bacteriology, 1961.). Mixtures of these or more complex natural mixtures, such as rumen fluid or Oxgall, can be used to induce germination. Oxgall is dehydrated bovine bile composed of fatty acids, bile acids, inorganic salts, sulfates, bile pigments, cholesterol, mucin, lecithin, glycuronic acids, porphyrins, and urea. The germination can also be performed in a growth medium like prereduced BHIS/oxgall germination medium, in which BHIS (Brain heart infusion powder (37 g/L), yeast extract (5 g/L), L-cysteine HCl (1 g/L)) provides peptides, amino acids, inorganic ions and sugars in the complex BHI and yeast extract mixtures and Oxgall provides additional bile acid germinants.

In addition, pressure may be used to germinate spores. The selection of germinants can vary with the microbe being sought. Different species require different germinants and different isolates of the same species can require different germinants for optimal germination. Finally, it is important to dilute the mixture prior to plating because some germinants are inhibitory to growth of the vegetative-state microorganisms. For instance, it has been shown that alkyl amines must be neutralized with anionic lipophiles in order to promote optimal growth. Bile acids can also inhibit growth of some organisms despite promoting their germination, and must be diluted away prior to plating for viable cells.

For example, BHIS/oxgall solution is used as a germinant and contains 0.5×BHIS medium with 0.25% oxgall (dehydrated bovine bile) where 1×BHIS medium contains the following per L of solution: 6 g Brain Heart Infusion from solids, 7 g peptic digest of animal tissue, 14.5 g of pancreatic digest of casein, 5 g of yeast extract, 5 g sodium chloride, 2 g glucose, 2.5 g disodium phosphate, and 1 g cysteine. Additionally, Ca-DPA is a germinant and contains 40 mM CaCl₂), and 40 mM dipicolinic acid (DPA). Rumen fluid (Bar Diamond, Inc.) is also a germinant.

Simulated gastric fluid (Ricca Chemical) is a germinant and is 0.2% (w/v) Sodium Chloride in 0.7% (v/v) Hydrochloric Acid. Mucin medium is a germinant and prepared by adding the following items to 1 L of distilled sterile water: 0.4 g KH₂PO₄, 0.53 g Na₂HPO₄, 0.3 g NH₄Cl, 0.3 g NaCl, 0.1 g MgCl₂×6H₂O, 0.11 g CaCl₂), 1 ml alkaline trace element solution, 1 ml acid trace element solution, 1 ml vitamin solution, 0.5 mg resazurin, 4 g NaHCO₃, 0.25 g Na₂S×9 H₂O. The trace element and vitamin solutions prepared as described previously (Stams et al., 1993). All compounds were autoclaved, except the vitamins, which were filter-sterilized. The basal medium was supplemented with 0.7% (v/v) clarified, sterile rumen fluid and 0.25% (v/v) commercial hog gastric mucin (Type III; Sigma), purified by ethanol precipitation as described previously (Miller & Hoskins, 1981). This medium is referred herein as mucin medium.

Fetal Bovine Serum (Gibco) can be used as a germinant and contains 5% FBS heat inactivated, in Phosphate Buffered Saline (PBS, Fisher Scientific) containing 0.137M Sodium Chloride, 0.0027M Potassium Chloride, 0.0119M Phosphate Buffer. Thioglycollate is a germinant as described previously (Kamiya et al Journal of Medical Microbiology 1989) and contains 0.25M (pH10) sodium thioglycollate. Dodecylamine solution containing 1 mM dodecylamine in PBS is a germinant. A sugar solution can be used as a germinant and contains 0.2% fructose, 0.2% glucose, and 0.2% mannitol. Amino acid solution can also be used as a germinant and contains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mM proline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine. A germinant mixture referred to herein as Germix 3 can be a germinant and contains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mM proline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine, 0.2% taurocholate, 0.2% fructose, 0.2% mannitol, 0.2% glucose, 1 mM inosine, 2.5 mM Ca-DPA, and 5 mM KCl. BHIS medium+DPA is a germinant mixture and contains BHIS medium and 2 mM Ca-DPA. Escherichia coli spent medium supernatant referred to herein as EcSN is a germinant and is prepared by growing E. coli MG1655 in SweetB/Fos inulin medium anaerobically for 48 hr, spinning down cells at 20,000 rcf for 20 minutes, collecting the supernatant and heating to 60 C for 40 min. Finally, the solution is filter sterilized and used as a germinant solution.

Example 14: Selection of Media for Growth

It is important to select appropriate media to support growth, including preferred carbon sources. For example, some organisms prefer complex sugars such as cellobiose over simple sugars. Examples of media used in the isolation of sporulating organisms include EYA, BHI, BHIS, and GAM (see below for complete names and references). Multiple dilutions are plated out to ensure that some plates will have well isolated colonies on them for analysis, or alternatively plates with dense colonies may scraped and suspended in PBS to generate a mixed diverse community.

Plates are incubated anaerobically or aerobically at 37 C for 48-72 or more hours, targeting anaerobic or aerobic spore formers, respectively.

Solid plate media include:

-   -   Gifu Anaerobic Medium (GAM, Nissui) without dextrose         supplemented with fructooligosaccharides/inulin (0.4%), mannitol         (0.4%), inulin (0.4%), or fructose (0.4%), or a combination         thereof.     -   Sweet GAM [Gifu Anaerobic Medium (GAM, Nissui)] modified,         supplemented with glucose, cellobiose, maltose, L-arabinose,         fructose, fructooligosaccharides/inulin, mannitol and sodium         lactate)     -   Brucella Blood Agar (BBA, Atlas, Handbook of Microbiological         Media, 4th ed, ASM Press, 2010)     -   PEA sheep blood (Anaerobe Systems; 5% Sheep Blood Agar with         Phenylethyl Alcohol)     -   Egg Yolk Agar (EYA) (Atlas, Handbook of Microbiological Media,         4th ed, ASM Press, 2010)     -   Sulfite polymyxin milk agar (Mevissen-Verhage et al., J. Clin.         Microbiol. 25:285-289 (1987))     -   Mucin agar (Derrien et al., IJSEM 54: 1469-1476 (2004))     -   Polygalacturonate agar (Jensen & Canale-Parola, Appl. Environ.         Microbiol. 52:880-997 (1986))     -   M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM         Press, 2010)     -   M2 agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM         Press, 2010) supplemented with starch (1%), mannitol (0.4%),         lactate (1.5 g/L) or lactose (0.4%)     -   Sweet B—Brain Heart Infusion agar (Atlas, Handbook of         Microbiological

Media, 4th ed, ASM Press, 2010) supplemented with yeast extract (0.5%), hemin, cysteine (0.1%), maltose (0.1%), cellobiose (0.1%), soluble starch (sigma, 1%), MOPS (50 mM, pH 7).

-   -   PY-salicin (peptone-yeast extract agar supplemented with         salicin) (Atlas, Handbook of Microbiological Media, 4th ed, ASM         Press, 2010).     -   Modified Brain Heart Infusion (M-BHI) [[sweet and sour]]         contains the following per L: 37.5 g Brain Heart Infusion powder         (Remel), 5 g yeast extract, 2.2 g meat extract, 1.2 g liver         extract, 1 g cystein HCl, 0.3 g sodium thioglycolate, 10 mg         hemin, 2 g soluble starch, 2 g FOS/Inulin, 1 g cellobiose, 1 g         L-arabinose, 1 g mannitol, 1 Na-lactate, 1 mL Tween 80, 0.6 g         MgSO4×7H2O, 0.6 g CaCl₂), 6 g (NH4)2SO4, 3 g KH2PO4, 0.5 g         K2HPO4, 33 mM Acetic acid, 9 mM propionic acid, 1 mM Isobutyric         acid, 1 mM isovaleric acid, 15 g agar, and after autoclaving add         50 mL of 8% NaHCO₃ solution and 50 mL 1M MOPS-KOH (pH 7).     -   Noack-Blaut Eubacterium agar (See Noack et al. J. Nutr. (1998)         128:1385-1391)     -   BHIS az1/ge2—BHIS az/ge agar (Reeves et. al. Infect. Immun.         80:3786-3794 (2012)) [Brain Heart Infusion agar (Atlas, Handbook         of Microbiological Media, 4th ed, ASM Press, 2010) supplemented         with yeast extract 0.5%, cysteine 0.1%, 0.1% cellobiose, 0.1%         inulin, 0.1% maltose, aztreonam 1 mg/L, gentamycin 2 mg/L]     -   BHIS CInM az1/ge2—BHIS CInM [Brain Heart Infusion agar (Atlas,         Handbook of Microbiological Media, 4th ed, ASM Press, 2010)         supplemented with yeast extract 0.5%, cysteine 0.1%, 0.1%         cellobiose, 0.1% inulin, 0.1% maltose, aztreonam 1 mg/L,         gentamycin 2 mg/L]

Example 15: The Purification and Isolation of a Spore Forming Fraction from Feces

To purify and selectively isolate efficacious spores from fecal material a donation is first blended with saline using a homogenization device (e.g., laboratory blender) to produce a 20% slurry (w/v). 100% ethanol is added for an inactivation treatment that lasts 10 seconds to 1 hour. The final alcohol concentration can range from 30-90%, preferably 50-70%. High speed centrifugation (3200 rcf for 10 min) is performed to remove solvent and the pellet is retained and washed. Subsequently, once the washed pellet is resuspended, a low speed centrifugation step (200 rcf for 4 min) is performed to remove large particulate vegetative matter and the supernatant containing the spores is retained. High speed centrifugation (3200 rcf for 10 min) is performed on the supernatant to concentrate the spore material. The pellet is then washed and resuspended to generate a 20% slurry. This is the ethanol treated spore preparation. The concentrated slurry is then separated with a density based gradient e.g. a CsCl gradient, sucrose gradient or combination of the two generating a ethanol treated, gradient-purified spore preparation. For example, a CsCl gradient is performed by loading a 20% volume of spore suspension on top a 80% volume of a stepwise CsCl gradient (w/v) containing the steps of 64%, 50%, 40% CsCl (w/v) and centrifuging for 20 min at 3200 rcf. The spore fraction is then run on a sucrose step gradient with steps of 67%, 50%, 40%, and 30% (w/v). When centrifuged in a swinging bucket rotor for 10 min at 3200 rcf. The spores run roughly in the 30% and 40% sucrose fractions. The lower spore fraction (FIG. 2) is then removed and washed to produce a concentrated ethanol treated, gradient-purified spore preparation. Taking advantage of the refractive properties of spores observed by phase contrast microscopy (spores are bright and refractive while germinated spores and vegetative cells are dark) one can see an enrichment of the spore fraction from a fecal bacterial cell suspension (FIG. 3, left) compared to an ethanol treated, CsCl gradient purified, spore preparation (FIG. 3, center), and to an ethanol treated, CsCl gradient purified, sucrose gradient purified, spore preparation (FIG. 3, right).

Furthermore, growth of spores after treatment with a germinant can also be used to quantify a viable spore population. Briefly, samples were incubated with a germinant (Oxgall, 0.25% for up to 1 hour), diluted and plated anaerobically on BBA (Brucella Blood Agar) or similar media (e.g. see Examples 4 and 5). Individual colonies were picked and DNA isolated for full-length 16S sequencing to identify the species composition (e.g. see examples 2 and 3). Analysis revealed that 22 species were observed in total (Table 2) with a vast majority present in both the material purified with the gradient and without the gradient, indicating no or inconsequential shift in the ecology as a result of gradient purification. Spore yield calculations demonstrate an efficient recovery of 38% of the spores from the initial fecal material as measured by germination and plating of spores on BBA or measuring DPA count in the sample.

Example 16: Bacterial Compositions Prevent C. difficile Infection in a Mouse Model

To test the therapeutic potential of the bacterial compositions a prophylactic mouse model of C. difficile infection (model based on Chen, et al., A mouse model of Clostridium difficile associated disease, Gastroenterology 135(6):1984-1992) was used. Two cages of five mice each were tested for each arm of the experiment. All mice received an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and Vancomycin (0.056 mg/ml) in their drinking water on days −14 through −5 and a dose of 10 mg/kg Clindamycin by oral gavage on day −3. On day −1, they received either the test article or vehicle control via oral gavage. On day 0 they were challenged by administration of approximately 4.5 log 10 cfu of C. difficile (ATCC 43255) via oral gavage. Optionally a positive control group received vancomycin from day −1 through day 3 in addition to the antibiotic protocol and C. difficile challenge specified above. Feces were collected from the cages for analysis of bacterial carriage, mortality was assessed every day from day 0 to day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the vehicle were used to assess the success of the test article. Additionally, a C. difficile symptom scoring was performed each day from day −1 through day 6. Clinical Score was based on a 0-4 scale by combining scores for Appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), and Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).

In a naive control arm, animals were challenged with C. difficile. In the vancomycin positive control arm animals were dosed with C. difficile and treated with vancomycin from day −1 through day 3. The negative control was gavaged with PBS alone and no bacteria. The test arms of the experiment tested 1×, 0.1×, 0.01× dilutions derived from a single donor preparation of ethanol treated spores (e.g. see example 6) or the heat treated feces prepared by treating a 20% slurry for 30 min at 80 C. Dosing for CFU counts was determined from the final ethanol treated spores and dilutions of total spores were administered at 1×, 0.1×, 0.01× of the spore mixture for the ethanol treated fraction and a 1× dose for the heat treated fraction.

Weight loss and mortality were assessed on day 3. The negative control, treated with C. difficile only, exhibits 20% mortality and weight loss on Day 3, while the positive control of 10% human fecal suspension displays no mortality or weight loss on Day 3 (Table 3). EtOH-treated feces prevents mortality and weight loss at three dilutions, while the heat-treated fraction was protective at the only dose tested. These data indicate that the spore fraction is efficacious in preventing C. difficile infection in the mouse.

Example 17: The Prophylactic and Relapse Prevention Hamster Models

Previous studies with hamsters using toxigenic and nontoxigenic strains of C. difficile demonstrated the utility of the hamster model in examining relapse post antibiotic treatment and the effects of prophylaxis treatments with cecal flora in C. difficile infection (Wilson et al. 1981, Wilson et al. 1983, Borriello et al. 1985) and more broadly gastrointestinal infectious disease. To demonstrate prophylactic use of a test article to ameliorate C. difficile infection, the following hamster model is used. In a prophylactic model, Clindamycin (10 mg/kg s.c.) is given on day −5, the test article or control is administered on day −3, and C. difficile challenge occurs on day 0. In the positive control arm, vancomycin is then administered on day 1-5 (and vehicle control is delivered on day −3). Feces are collected on day −5, −4, −1, 1, 3, 5, 7, 9 and fecal samples are assessed for pathogen carriage and reduction by microbiological methods, 16S sequencing approaches or other methods utilized by one skilled in the art. Mortality is assessed throughout the experiment through 21 days post C. difficile challenge. The percentage survival curves show that ethanol treated spores and ethanol treated, gradient-purified spores better protect the hamsters compared to the Vancomycin control, and vehicle control.

See FIG. 4: Prophylaxis model with the ethanol treated spore preparation and the ethanol treated, gradient-purified spore preparation.

In the relapse prevention model, hamsters are challenged with toxigenic C. difficile strains on day 0, and treated with clindamycin by oral gavage on day 1, and vancomycin dosing day 2-6. Test or control treatment was then administered on day 7, 8, and 9. The groups of hamsters for each arm consist of 8 hamsters per group. Fecal material is collected on day −1, 1, 3, 5, 7, 10 and 13 and hamster mortality is assessed throughout. Survival curves are used to assess the success of the test article e.g. ethanol treated or ethanol treated, gradient purified spores versus the control treatment in preventing hamster death. The survival curves demonstrate maximum efficacy for the ethanol treated, gradient-purified spores followed by the ethanol treated spores. Both treatments improved survival percentage over vancomycin treatment alone.

See FIG. 5: Relapse prevention model with ethanol treated spores and ethanol treated, gradient purified spores

Example 18: Clinical Treatment of Recurrent C. difficile in Patients

To assess the efficacy of a test article (e.g., ethanol treated spore preparations, see Example 15) to treat recurrent C. difficile in human patients, the following procedure was performed to take feces from a healthy donor, inactivate via the ethanol treated spore preparation protocol described below, and treat recurrent C. difficile in patients presenting with this indication. Non-related donors were screened for general health history for absence of chronic medical conditions (including inflammatory bowel disease; irritable bowel syndrome; Celiac disease; or any history of gastrointestinal malignancy or polyposis), absence of risk factors for transmissible infections, antibiotic non-use in the previous 6 months, and negative results in laboratory assays for blood-borne pathogens (HIV, HTLV, HCV, HBV, CMV, HAV and Treponema pallidum) and fecal bacterial pathogens (Salmonella, Shigella, Yersinia, Campylobacter, E. coli 0157), ova and parasites, and other infectious agents (Giardia, Cryptosporidium Cyclospora, Isospora) prior to stool donation.

Donor stool was frozen shortly after donation and sampled for testing. At the time of use, approximately 75 g of donor stool was thawed and resuspended in 500 mL of non-bacteriostatic normal saline and mixed in a single use glass or plastic blender. The resulting slurry was sequentially passed through sterile, disposable mesh screens that remove particles of size 600, 300 and 200 microns. The slurry was then centrifuged briefly (200 rcf for 4 min) to separate fibrous and particulate materials, and the supernatant (containing bacterial cells and spores) was transferred to a fresh container. Ethanol was added to a final concentration of 50% and the resulting ˜1500 ml slurry was incubated at room temperature for 1 hr with continuous mixing to inactivate vegetative bacterial cells. Midway through inactivation the slurry was transferred to a new bottle to ensure complete contact with the ethanol. The solid matter was pelleted in a centrifuge and washed 3 times with normal saline to remove residual ethanol. The final pellet was resuspended in 100% sterile, USP glycerol at a minimum volume, and filled into approximately 30 size 0 delayed release capsules (hypromellose DRcaps, Capsugel, Inc.) at 0.65 mL suspension each. The capsules were immediately capped and placed onto an aluminum freezing block held at −80° C. via dry ice to freeze. The frozen capsules were in turn over-capsulated with size 00 DRcaps to enhance capsule stability, labeled, and placed into <−65° C. storage immediately. The final product was stored at <−65° C. until the day and time of use. Encapsulated product may be stored for indefinitely at <−65° C. On the day of dosing capsules were warmed on wet ice for 1 to 2 hours to improve tolerability, and were then dosed with water ad libitium.

Patient 1 is a 45-year old woman with a history of C. difficile infection and diarrhea for at least 1 year prior to treatment. She has been previously treated with multiple courses of antibiotics followed each time by recurrence of C. difficile-associated diarrhea.

Patient 2 is an 81-year old female who has experienced recurrent C. difficile infection for 6 months prior to treatment despite adequate antibiotic therapy following each recurrence.

24 hours prior to starting oral treatment, CDAD antibiotic therapy was discontinued. Each patient received a colon preparation procedure intended to reduce the competing microbial burden in the gastrointestinal tract and to facilitate repopulation by the spore forming organisms in the investigational product.

On the morning of the first treatment day, the patients received a dose of delayed release capsules containing the investigational product with water ad libitum. Patients were requested to avoid food for 1 hour thereafter. The next day, the patient returned to the clinic to receive an additional dose. Patients were asked to avoid food for 4 hours prior to receiving their second dose and for 1 hour following dosing.

Both patients were followed closely for evidence of relapse or adverse symptoms following treatment. Patients were contacted by phone on Day 2, Day 4, and Weeks 1, 2 and 4 and each was queried about her general status and the condition of her CDAD and related symptoms. Stool samples were collected at baseline and Weeks 1, 2, 4 and 8 post-treatment to assess changes in the gut microbiota via 16S sequencing and spore count with methods explained previously (e.g. see Examples 11 and 12). Through 4 weeks post treatment, each patient has gradually improved with no evidence of C. difficile recurrence.

Six other patients with recurrent C. difficile-associated diarrhea were treated in a similar fashion, with no CDI recurrence and no requirement for resumption of antibiotics (total of 8 patients). Additionally, there were no treatment-related serious adverse events.

Example 19: Treatment of Fecal Suspensions with Ethanol or Heat Drastically Reduces Vegetative Cell Numbers and Results in an Enrichment of Spore Forming Species

Treatment of a sample, preferably a human fecal sample, in a manner to inactivate or kill substantially all of the vegetative forms of bacteria present in the sample results in selection and enrichment of the spore fraction. Methods for inactivation can include heating, sonication, detergent lysis, enzymatic digestion (such as lysozyme and/or proteinase K), ethanol or acid treatment, exposure to solvents (Tetrahydrofuran, 1-butanol, 2-butanol, 1,2 propanediol, 1,3 propanediol, butanoate, propanoate, chloroform, dimethyl ether and a detergent like triton X-100, diethyl ether), or a combination of these methods. To demonstrate the efficacy of ethanol induced inactivation of vegetative cells, a 10% fecal suspension was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 min. The suspension was incubated at room temperature for 30 min, 1 h, 4 h or 24 h. After incubation the suspension was centrifuged at 13,000 rpm for 5 min to pellet spores. The supernatant is discarded and the pellet is resuspended in equal volume of PBS. Viable cells were measured as described below.

To demonstrate the efficacy of heat treatment on vegetative cell inactivation a 10-20% fecal suspension was incubated at 70 C, 80 C, 90 C or 100 C for 10 min or 1 h.

After ethanol or heat treatment, remaining viable cells were measured after 24 h incubation on plates by determining the bacterial titer on Brucella blood agar (BBA) as a function of treatment and time (See FIG. 6). Ethanol treatment for 1 h and 25 h have similar effects, reducing the number of viable cells by approximately 4 logs, while increasing temperature and time at high temperature leads to higher losses in viable cell number, with no colonies detectable at 100° C. at either 10 min or 1 h. In this experiment no germinants were used. After several days of additional growth on plates, a number of colonies were picked from these treated samples and identified by 16S rDNA analysis (e.g. see Examples 11 and 12). These included known spore forming Clostridium spp. as well as species not previously reported to be spore formers including Ruminococcus bromii, and Anaerotruncus colihominis (Lawson, et al 2004), and a Eubacterium sp. (Table 4). See FIG. 6: Heat and ethanol treatments reduce cell viability

To demonstrate that vegetative cells are greatly reduced by ethanol treatment, known non-spore forming bacteria are ethanol treated as described previously (e.g. see Example 15) and viability was determined by plating on BBA in anaerobic conditions (e.g. see Example 14). Fecal material from four independent donors was exposed to 60 C for 5 min and subsequently plated on three types of selective media under either aerobic (+O₂) or anaerobic conditions (−O₂) (BBA+aerobic, MacConkey lactose+aerobic, Bacteroides Bile esculin+anaerobic) to identify known nonsporeforming Enterobacteria (survivors on MacConkey agar) and Bacteroides fragilis group species (survivors on Bacteroides Bile Esculin plates). The detectable limit for these assays was roughly 20 cfu/m L. Germinants were not used in this experiment (FIG. 7). Both ethanol and heat inactivation greatly reduces the cell viability from fecal material to the limit of detection under using MacConkey lactose agar and BBE agar. The remaining cells identified on BBA media grown in anaerobic conditions comprise the non-germinant dependent spore forming species. See FIG. 7: Reduction in non-spore forming vegetative cells by treatment at 60° C. for 5 min

Additionally, the ethanol treatment was shown to rapidly kill both aerobic and non-spore forming anaerobic colony forming units in 10% fecal suspensions as determined by plating on rich (BBA) media. The reduction of plated CFUs decreases four orders of magnitude in seconds as shown in FIG. 8.

See FIG. 8: Time course demonstrates ethanol reduces both anaerobic and aerobic bacterial CFUs

Example 20: Species Identified and Isolated as Spore Formers by Ethanol Treatment

To demonstrate that spore-forming species are enriched by heat or ethanol treatment methods, a comparison of >7000 colony isolates was performed to identify species in a repeatable fashion (e.g., identified independently in multiple preparations, see examples 1, 2, and 3) only isolated from fecal suspensions treated with 50% ethanol or heat treatment and not from untreated fecal suspensions (Table 5). These data demonstrate the ability to select for spore forming species from fecal material, and identify organisms as spore formers not previously described as such in the literature. In each case, organisms were picked as an isolated colony, grown anaerobically, and then subjected to full-length 16S sequencing in order to assign species identity.

To further identify spore formers, ethanol treated fecal samples from donors A, B, C, D, E and F were plated to a variety of solid media types, single colonies were picked and grown up in broth in a 96 well format (Table 6-11). The 16S rRNA gene was then amplified by PCR and direct cycle sequencing was performed (See examples 11 and 12). The ID is based on the forward read from direct cycle sequencing of the 16S rRNA gene.

There is surprising heterogeneity in the microbiome from one individual to another (Clemente et al., 2012) and this has consequences for determining the potential efficacy of various donors to generate useful spore compositions. The method described below is useful for screening donors when, for instance, a particular quantity or diversity of spore forming organisms is useful or desired for repopulating the microbiome following antibiotic treatment or treating a particular disease or condition. Further, such screening is useful when there is a need to screen donors for the purpose of isolating microorganisms capable of spore formation, or when a purified preparation of spore forming organisms is desired from a particular donor.

Total spore count is also a measure of potency of a particular donation or purified spore preparation and is vital to determine the quantity of material required to achieve a desired dose level. To understand the variability in total spore counts, donor samples were collected and processed as described in prior examples. Donor spore counts in CFU/g were then determined by growth on media plates at various titrations to determine the spore content of the donation. Furthermore, DPA assays were used to assess spore content (expressed as spore equivalents) as described in Example 21. As seen in FIG. 9, there is as much as two logs difference in an individual donor over time and can be up to three logs difference between donors. One possible reason for the difference in spore content measures is that nonviable spores and non-germinable spores will not be observed by plating but will have measurable DPA content. Another possibility is the variability between species of DPA content in spores making some complex mixtures containing high DPA spores while other mixtures contain low DPA content spores. Selecting donors with high spore counts is important in determining productivity of isolating spores from fecal donations by identifying preferred donors.

See FIG. 9: Donation Spore concentrations from clinical donors

A fresh fecal sample from donor F was treated as described in Example 15 to generate an ethanol treated spore fraction, germinated with BHIS/Oxgall for 1 h as a described (e.g. see Example 13), then plated to a variety of media (e.g. See example 14). Colonies were picked with a focus on picking several of each type of morphologically distinct colony on each plate to capture as much diversity as possible. Colonies are counted on a plate of each media type with well isolated colonies such that the number of colony forming units per ml can be calculated (Table 12). Colonies were picked into one of several liquid media and the 16S rDNA sequences (e.g. see Examples 11 and 12) were determined and analyzed as described above. The number of unique OTUs for each media type is shown below with the media with the most unique OTUs at the top (Table 12). Combinations of 3 to 5 of the top 5 media types capture diversity, and some other can be chosen to target specific species of interest. Colony forming units can be calculated for a given species using the 16S data, and could be used to determine whether a sufficient level of a given organism is present. The spore complement from Donor F as determined in this experiment includes these 52 species as determined by 16S sequencing (Table 12).

To screen human donors for the presence of a diversity of spore forming bacteria and/or for specific spore-forming bacteria, fecal samples were prepared using germinants and selective plating conditions and colonies were picked (e.g. see Examples 13 and 14) and analyzed for 16S diversity as described previously (see Examples 11 and 12). An assessment of donor diversity could include the cfu/ml of ethanol resistant cells on a given media type, or cfu/ml of a given species using the 16S analysis of colonies picked from that media to determine the level of spores of a given species of interest. This type of culture-based analysis could be complemented by culture-independent methods such as qPCR with probes specific to species or genera of interest or metagenomic sequencing of spore preparations, or 16S profiling of spore preparations using IIlumina 16S variable region sequencing approaches (e.g. see Examples 11 and 12).

Example 21: Quantification of Spore Concentrations Using DPA Assay

Methods to assess spore concentration in complex mixtures typically require the separation and selection of spores and subsequent growth of individual species to determine the colony forming units. The art does not teach how to quantitatively germinate all the spores in a complex mixture as there are many species for which appropriate germinants have not been identified. Furthermore, sporulation is thought to be a stochastic process as a result of evolutionary selection, meaning that not all spores from a single species germinate with same response to germinant concentration, time and other environmental conditions. Alternatively, a key metabolite of bacterial spores, dipicolinic acid (DPA) has been developed to quantify spores particles in a sample and avoid interference from fecal contaminants. The assay utilizes the fact that DPA chelates Terbium 3+ to form a luminescent complex (Fichtel et al, FEMS Microbiology Ecology, 2007; Kort et al, Applied and Environmental Microbiology, 2005; Shafaat and Ponce, Applied and Environmental Microbiology, 2006; Yang and Ponce, International Journal of Food Microbiology, 2009; Hindle and Hall, Analyst, 1999). A time-resolved fluorescence assay detects terbium luminescence in the presence of DPA giving a quantitative measurement of DPA concentration in a solution.

To perform the assay 1 mL of the spore standard to be measured was transferred to a 2 mL microcentrifuge tube. The samples were centrifuged at 13000 RCF for 10 min and the sample is washed in 1 mL sterile deionized H₂O. Wash an additional time by repeating the centrifugation. Transfer the 1 mL solution to hungate tubes and autoclave samples on a steam cycle for 30 min at 250 C. Add 100 uL of 30 uM TbCl₃ solution (400 mM sodium acetate, pH 5.0, 30 μM TbCl₃) to the sample. Make serial dilutions of of the autoclaved material and measure the fluorescence of each sample by exciting with 275 nm light and measuring the emission wavelength of 543 nm for an integration time of 1.25 ms and a 0.1 ms delay.

Purified spores are produced as described previously (e.g. see www.epa.gov/pesticides/methods/MB-28-00.pdf). Serial dilutions of purified spores from C. bifermentans, C. sporogenes, and C. butyricum cultures were prepared and measured by plating on BBA media and incubating overnight at 37 C to determine CFU/ml. FIG. 10 shows the linear correspondence across different spore producing bacteria across several logs demonstrating the DPA assay as means to assess spore content.

See FIG. 10: Linear range of DPA assay compared to CFU counts/ml

The discrepancy for complex spore populations between spore counts measured by germinable spore CFU and by DPA has important implications for determining the potency of an ethanol treated spore preparation for clinical use. Table AC shows spore content data from 3 different ethanol treated spore preparations used to successfully treat 3 patients suffering from recurrent C. difficile infection. The spore content of each spore preparation is characterized using the two described methods.

TABLE AC Spore quantitation for ethanol treated spore preparations using spore CFU (SCFU) assay and DPA assay SCFU/30 DPA SEq/30 Ratio SCFU/ Preparation capsules capsules DPA Preparation 1 4.0 × 10{circumflex over ( )}5 6.8 × 10{circumflex over ( )}7 5.9 × 10−3 Preparation 2 2.1 × 10{circumflex over ( )}7 9.2 × 10{circumflex over ( )}8 0.023 Preparation 3 6.9 × 10{circumflex over ( )}9 9.6 × 10{circumflex over ( )}9 0.72

What is immediately apparent is that spore content varies greatly per 30 capsules. As measured by germinable SCFU, spore content varies by greater than 10,000-fold. As measured by DPA, spore content varies by greater than 100-fold. In the absence of the DPA assay, it would be difficult to set a minimum dose for administration to a patient. For instance, without data from the DPA assay, one would conclude that a minimum effective dose of spores is 4×10⁵ or less using the SCFU assay (e.g. Preparation 1, Table AC). If that SCFU dose was used to normalize dosing in a clinical setting, however, then the actual spore doses given to patients would be much lower for other ethanol treated spore preparations as measured as by the DPA assay (Table AD).

TABLE AD DPA doses in Table AC when normalized to 4 × 10⁵ SCFU per dose SCFU/30 DPA SEq/30 Fraction of Preparation capsules capsules Preparation 1 Dose Preparation 1 4.0 × 10{circumflex over ( )}5 6.8 × 10{circumflex over ( )}7 1.0 Preparation 2 4.0 × 10{circumflex over ( )}5 1.8 × 10{circumflex over ( )}7 0.26 Preparation 3 4.0 × 10{circumflex over ( )}5 5.6 × 10{circumflex over ( )}5 0.0082

It becomes clear from the variability of SCFU and DPA counts across various donations that using SCFU as the measure of potency would lead to significant underdosing in certain cases. For instance, setting a dose specification of 4×10⁵ SCFU (the apparent effective dose from donor Preparation 1) for product Preparation 3 would lead to a potential underdosing of more than 100-fold. This can be rectified only by setting potency specifications based on the DPA assay, which better reflects total spore counts in an ethanol treated spore preparation. The unexpected finding of this work is that the DPA assay is uniquely suited to set potency and determine dosing for an ethanol treated spore preparation.

Example 22: Demonstration of Enhanced Growth with a Germinant

To enhance the ethanol treated spores germination capability and demonstrate spore viability, spores from three different donors were germinated by various treatments and plated on various media. Germination with BHIS/oxgall (BHIS ox), Ca-DPA, rumen fluid (RF), simulated gastric fluid (SGF), mucin medium (Muc), fetal bovine serum (FBS), or thioglycollate (Thi) for 1 hour at 37 C in anaerobic conditions was performed as described previously (e.g. see Examples 13 and 14) with samples derived from two independent donors (FIG. 11). The spore-germinant mixture was serially diluted and plated on different plate media including BBA, Sweet B, Sweet B+lysozyme (2 ug/ml), M2GSC and M2GSC+lysozyme (2 ug/ml) as previously described (e.g. see Examples 13 and 14) to determine spore germination. Colony forming units were tallied and titers were determined using standard techniques by one skilled in the art. As FIG. 11 shows, maximum colony forming units are derived from BHI-oxgall treatment. This germination treatment also greatly increases the diversity as measured by the number of OTUs identified when samples were submitted for 16S sequencing (e.g. see Examples 11 and 12) compared to plating without a germination step (FIG. 12). As shown in FIG. 11: Different germinant treatments have variable effects on CFU counts from donor A (upper left) and donor B (lower right). The Y-Axes are spore CFU per ml. As shown in FIG. 12: Germinates greatly increase the diversity of cultured spore forming OTUs.

To test the effect of heat activation to promote germination, ethanol treated fecal samples were treated for 15 min at room temperature, 55 C, 65 C, 75 C or 85 C from three different donors and germinated subsequently with BHIS+Oxgall for 1 hr at 37 C then plated on BBA media (FIG. 13) as previously described (e.g. see Examples 13 and 14). Pretreatment at room temperature produced equal if not more spores than the elevated temperatures in all three donors. The temperature of germinating was also examined by incubating samples at room temperature or 37 C for 1 hr in anaerobic conditions before plating on BBA. No difference in the number of CFUs was observed between the two conditions. Lysozyme addition to the plates (2 ug/ml) was also tested on a single donor sample by the testing of various activation temperature followed by an incubation in the presence or absence of lysozyme. The addition of lysozyme had a small effect when plated on Sweet B or M2GSC media but less so than treatment with BHIS oxgall without lysozyme for 1 hr (FIG. 14).

As shown in FIG. 13: Heat Activation as a germination treatment with BHIS+oxgall. As shown in FIG. 14: Effect of lysozyme slightly enhances germination.

Germination time was also tested by treating a 10% suspension of a single donor ethanol treated feces (e.g. see Example 15) incubated in either BHIS, taurocholate, oxgall, or germ ix for 0, 15, 30, or 60 minutes and subsequently plated on BHIS, EYA, or BBA media (e.g. see Examples 13 and 14). 60 minutes resulted in the most CFU units across all various combinations germinates and plate media tested.

Example 23: Demonstrating Efficacy of Germinable and Sporulatable Fractions of Ethanol Treated Spores

To define methods for characterization and purification, and to improve (e.g., such as by modulating the diversity of the compositions) the active spore forming ecology derived from fecal donations, the ethanol treated spore population (as described in Example 15) was further fractionated. A “germinable fraction” was derived by treating the ethanol-treated spore preparation with oxgall, plating to various solid media, and then, after 2 days or 7 days of growth, scraping all the bacterial growth from the plates into 5 mL of PBS per plate to generate a bacterial suspension. A “sporulatable fraction” was derived as above except that the cells were allowed to grow on solid media for 2 days or 7 days (the time was extended to allow sporulation, as is typical in sporulation protocols), and the resulting bacterial suspension was treated with 50% ethanol to derive a population of “sporulatable” spores, or species that were capable of forming spores. In preparing these fractions, fecal material from donor A was used to generate an ethanol treated spore preparation as previously described in Example 15; then spore content was determined by DPA assay and CFU/ml grown on various media (FIG. 15) as previously described (see Example 21). See FIG. 15: Spores initially present in ethanol treated spore preparation as measured by DPA and CFU/ml grown on specified media.

To characterize the fraction that is sporulatable, the 2 day and 7 day “germinable” fractions were assessed for CFU and DPA content before and after ethanol treatment to generate a spore fraction. Bacterial suspensions were treated with ethanol, germinated with Oxgall, and plated on the same types of media that the “germinable” fraction was grown on. DPA data showed that growth on plates for 2 and 7 days produced the same amount of total spores. Colonies on the several types of media were picked for 16S sequence analysis to identify the spore forming bacteria present (Table 13).

A 2 day “germinable” fraction and a 7 day “sporulatable” fraction were used as a treatment in the mouse prophylaxis assay as described (e.g. see Example 16). As a control, a 10% fecal suspension prepared from a donor (Donor B) was also administered to mice to model fecal microbiota transplant (FMT). Weight loss and mortality of the various test and control arms of the study are plotted in FIG. 17 and summarized in Table 15 which also contains the dosing information. The data clearly shows both the “germinable” and “sporulatable” fractions are efficacious in providing protection against C. difficile challenge in a prophylaxis mouse model (e.g. see Example 16). The efficacy of these fractions further demonstrates that the species present are responsible for the efficacy of the spore fraction, as the fractionation further dilutes any potential contaminant from the original spore preparation.

See FIG. 16: Titer of “germinable” fraction after 2 days (left) and Sporulatable fraction (right) by DPA and CFU/ml. The “sporulatable” fraction made following 7 days of growth was measured as previously described using germination and growth assays or DPA content as previously described (see Example 21).

The species present in the “germinable” and “sporulatable” fractions were determined by full length 16S sequencing of colony picks and by 16S NGS sequencing of the fractions themselves. The colony pick data indicate Clostridium species are very abundant in both fractions, while the NGS data reveal other spore forming organisms that are typically found in ethanol treated spore preparations are present.

Results are shown in the following: See Table 13. Species identified as “germinable” and “sporulatable” by colony picking approach. See Table YYY. Species identified as “germinable” using 16S-V4 NGS approach. See Table ZZZ. Species identified as “sporulatable” using 16s-V4 NGS approach. See FIG. 17: Mouse prophylaxis model demonstrates “germinable” and “sporulatable” preparations are protective against C. difficile challenge. Each plot tracks the change in the individual mouse's weight relative to day −1 over the course of the experiment. The number of deaths over the course of the experiment is indicated at the top of the chart and demonstrated by a line termination prior to day 6. The top panels (from left to right) are the vehicle control arm, the fecal suspension arm, and the untreated naive control arm, while the bottom panels are the ethanol treated, gradient purified spore preparation; the ethanol treated, gradient purified, “germinable” preparation, and ethanol treated, gradient purified, “sporulatable” preparation. See Table 15: Results of the prophylaxis mouse model and dosing information

Example 24: Donor Pooling Efficacy in Prophylaxis Mouse

To test the efficacy and dosing of pooled donor samples the C. difficile prophylaxis mouse model (e.g., see Example 16) is used with donations mixed from two or more donor samples as previously described. Weight loss and mortality with the mixed spore product versus the spore product derived from a single donor at the various dosing is determine whether the two treatment schemes are equivalent or one is significantly better than the other.

Dosing of the spore product derived from a single or multiple donors is between 1E4 to 1E10 CFU/ml. The spore product is mixed from product derived from any number of donors ranging from 1-10 at either equal concentrations or different known concentrations.

Additionally, this method can be used to expand the spore fraction for production purposes. For production purposes, an enriched spore fraction (e.g.—a purified and EtOH treated fraction of a fecal sample) is preserved in multiple aliquots to form a bank of viable spore-forming organisms. An aliquot of this bank is then recovered by germinant treatment followed by cultivation in a medium, and under conditions, that are broadly permissive for spore-forming organisms and encourage sporulation. After a suitable amplification time, the amplified bacteria including spores are harvested, and this preparation is solvent or heat-treated to isolate the spore fraction. This fraction may be further purified away from non-spore forms and culture constituents. The process of amplification, spore isolation and optional purification may be repeated at increasing scales to generate large quantities for further use. When enough germinable/sporulatable material has been accumulated by amplification, it may be further purified, concentrated or diluted, and/or preserved to a state suitable for further use, e.g.—clinical dosing.

Features may be incorporated into the above process to make it suitable for further utility, especially for product applications such as clinical use. The production of the initial spore fraction may be conducted under controlled conditions (cGMP's) and validated to remove non-spore organisms to a high degree. The germination may be conducted using reagents that are more standardizable than natural products such as oxgall, e.g.—synthetic mixtures of bile salts. Amplification may be done using media with components that are preferred for clinical safety, e.g.—sourced from qualified animals, or non-animal sourced. Conditions may be arranged so as to ensure consistent compositions of sporulated organisms, are less prone to contamination, and are more amenable to scale-up, e.g.—closed stirred fermenters with feedback control loops. Sporulated organisms from the process may be isolated using procedures that alone or combined stringently eliminate non-spores and other process residuals, e.g.—solvent treatment, aqueous two-phase extraction, and/or 60° C. long-time heat treatment. Preservation may involve addition of excipients and/or adjustment of conditions to enable conversion to a preferred dosage form amenable to long-term shelf storage, e.g.—addition of trehalose, followed by lyophilization or spray drying, further blending of the powder with microcrystalline cellulose, and encapsulation in a gelatin capsule to form an orally dosable product.

Example 25: Engraftment, Augmentation and Reduction of Pathogen Carriage in Patients Treated with Spore Compositions

Complementary genomic and microbiological methods were used to characterize the composition of the microbiota from Patient 1, 2, 3, 4, and 5, 6, 7, 8, 9, and 10 at pretreatment (pretreatment) and on up to 4 weeks post-treatment.

To determine the OTUs that engraft from treatment with an ethanol treated spore preparation in the patients and how their microbiome changed in response, the microbiome was characterized by 16S-V4 sequencing prior to treatment (pretreatment) with an ethanol treated spore preparation and up to 25 days after receiving treatment. As example, the treatment of patient 1 with an ethanol treated spore preparation led to the engraftment of OTUs from the spore treatment and augmentation in the microbiome of the patient (FIG. 18 and FIG. 19). By day 25 following treatment, the total microbial carriage was dominated by species of the following taxonomic groups: Bacteroides, Sutterella, Ruminococcus, Blautia, Eubacterium, Gemmiger/Faecalibacterium, and the non-sporeforming Lactobacillus (see Table 16 and Table 2 for specific OTUs). The first two genera represent OTUs that do not form spores while the latter taxonomic groups represent OTUs that are believed to form spores.

Patient treatment with the ethanol treated spore preparation leads to the establishment of a microbial ecology that has greater diversity than prior to treatment (FIG. 18). Genomic-based microbiome characterization confirmed engraftment of a range of OTUs that were absent in the patient pretreatment (Table 16). These OTUs comprised both bacterial species that were capable and not capable of forming spores, and OTUs that represent multiple phylogenetic clades. Organisms absent in Patient 1 pre-treatment either engraft directly from the ethanol treated spore fraction or are augmented by the creation of a gut environment favoring a healthy, diverse microbiota. Furthermore, Bacteroides fragilis group species were increased by 4 and 6 logs in patients 1 and 2 (FIG. 20).

OTUs that comprise an augmented ecology are not present in the patient prior to treatment and/or exist at extremely low frequencies such that they do not comprise a significant fraction of the total microbial carriage and are not detectable by genomic and/or microbiological assay methods. OTUs that are members of the engrafting and augmented ecologies were identified by characterizing the OTUs that increase in their relative abundance post treatment and that respectively are: (i) present in the ethanol treated spore preparation and absent in the patient pretreatment, or (ii) absent in the ethanol treated spore preparation, but increase in their relative abundance through time post treatment with the preparation due to the formation of favorable growth conditions by the treatment. Notably, the latter OTUs can grow from low frequency reservoirs in the patient, or be introduced from exogenous sources such as diet. OTUs that comprise a “core” augmented or engrafted ecology can be defined by the percentage of total patients in which they are observed to engraft and/or augment; the greater this percentage the more likely they are to be part of a core ecology responsible for catalyzing a shift away from a dysbiotic ecology. The dominant OTUs in an ecology can be identified using several methods including but not limited to defining the OTUs that have the greatest relative abundance in either the augmented or engrafted ecologies and defining a total relative abundance threshold. As example, the dominant OTUs in the augmented ecology of Patient-1 were identified by defining the OTUs with the greatest relative abundance, which together comprise 60% of the microbial carriage in this patient's augmented ecology.

See FIG. 18: Microbial diversity measured in the ethanol treated spore treatment sample and patient pre- and post-treatment samples. Total microbial diversity is defined using the Chao1 Alpha-Diversity Index and is measured at different genomic sampling depths to confirm adequate sequence coverage to assay the microbiome in the target samples. The patient pretreatment (purple) harbored a microbiome that was significantly reduced in total diversity as compared to the ethanol treated spore treatment (red) and patient post treatment at days 5 (blue), 14 (orange), and 25 (green).

See FIG. 19: Patient microbial ecology is shifted by treatment with an ethanol treated spore treatment from a dysbiotic state to a state of health. Principle Coordinates Analysis based on the total diversity and structure of the microbiome (Bray-Curtis Beta-Diversity) of the patient pre- and post-treatment delineates that the engraftment of OTUs from the spore treatment and the augmentation of the patient microbial ecology leads to a microbial ecology that is distinct from both the pretreatment microbiome and the ecology of the ethanol treated spore treatment (Table 16).

See FIG. 20: Augmentation of Bacteroides species in patients. Comparing the number of Bacteroides fragilis groups species per cfu/g of feces pre-treatment and in week 4 post treatment reveals an increase of 4 logs or greater. The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolution of the 16S-V4 region of the 16S gene for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation to a given sequence read. Given the topological nature of a phylogenetic tree and that the tree represents hierarchical relationships of OTUs to one another based on their sequence similarity and an underlying evolutionary model, taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1). Using this approach, clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood or other phylogenetic models familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another (generally, 1-5 bootstraps), and (ii) within a 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. The power of clade based analysis is that members of the same clade, due to their evolutionary relatedness, play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention.

Stool samples were aliquoted and resuspended 10×vol/wt in either 100% ethanol (for genomic characterization) or PBS containing 15% glycerol (for isolation of microbes) and then stored at −80° C. until needed for use. For genomic 16S sequence analysis colonies picked from plate isolates had their full-length 16S sequence characterized as described in Examples 11 and 12, and primary stool samples were prepared targeting the 16S-V4 region using the method for heterogeneous samples in Example 10.

Notably, 16S sequences of isolates of a given OTU are phylogenetically placed within their respective clades despite that the actual taxonomic assignment of species and genus may suggest they are taxonomically distinct from other members of the clades in which they fall. Discrepancies between taxonomic names given to an OTU is based on microbiological characteristics versus genetic sequencing are known to exist from the literature. The OTUs footnoted in this table are known to be discrepant between the different methods for assigning a taxonomic name.

Engraftment of OTUs from the ethanol treated spore preparation treatment into the patient as well as the resulting augmentation of the resident microbiome led to a significant decrease in and elimination of the carriage of pathogenic species other than C. difficile in the patient. 16S-V4 sequencing of primary stool samples demonstrated that at pretreatment, 20% of reads were from the genus Klebsiella and an additional 19% were assigned to the genus Fusobacterium. These striking data are evidence of a profoundly dysbiotic microbiota associated with recurrent C. difficile infection and chronic antibiotic use. In healthy individuals, Klebsiella is a resident of the human microbiome in only about 2% of subjects based on an analysis of HMP database (www.hmpdacc.org), and the mean relative abundance of Klebsiella is only about 0.09% in the stool of these people. It's surprising presence at 20% relative abundance in Patient 1 before treatment is an indicator of a proinflammatory gut environment enabling a “pathobiont” to overgrow and outcompete the commensal organisms normally found in the gut. Similarly, the dramatic overgrowth of Fusobacterium indicates a profoundly dysbiotic gut microbiota. One species of Fusobacterium, F. nucleatum (an OTU phylogenetically indistinguishable from Fusobacterium sp. 3_1_33 based on 16S-V4), has been termed “an emerging gut pathogen” based on its association with IBD, Crohn's disease, and colorectal cancer in humans and its demonstrated causative role in the development of colorectal cancer in animal models [Allen-Vercoe, Gut Microbes (2011) 2:294-8]. Importantly, neither Klebsiella nor Fusobacterium was detected in the 16S-V4 reads by Day 25 (Table 18).

To further characterize the colonization of the gut by Klebsiella and other Enterobacteriaceae and to speciate these organisms, pretreatment and Day 25 fecal samples stored at −80 C as PBS-glycerol suspensions were plated on a variety of selective media including MacConkey lactose media (selective for gram negative enterobacteria) and Simmons Citrate Inositol media (selective for Klebsiella spp) [Van Cregten et al, J. Clin. Microbiol. (1984) 20: 936-41]. Enterobacteria identified in the patient samples included K. pneumoniae, Klebsiella sp. Co_9935 and E. coli. Strikingly, each Klebsiella species was reduced by 2-4 logs whereas E. coli, a normal commensal organism present in a healthy microbiota, was reduced by less than 1 log (Table 19). This decrease in Klebsiella spp. carriage is consistent across multiple patients (Table 19). Four separate patients were evaluated for the presence of Klebsiella spp. pre treatment and 4 weeks post treatment. Klebsiella spp. were detected by growth on selective Simmons Citrate Inositol media as previously described. Serial dilution and plating, followed by determining cfu/mL titers of morphologically distinct species and 16S full length sequence identification of representatives of those distinct morphological classes, allowed calculation of titers of specific species.

The genus Bacteroides is an important member of the gastrointestinal microbiota; 100% of stool samples from the Human Microbiome Project contain at least one species of Bacteroides with total relative abundance in these samples ranging from 0.96% to 93.92% with a median relative abundance of 52.67% (www.hmpdacc.org reference data set HMSMCP). Bacteroides in the gut has been associated with amino acid fermentation and degradation of complex polysaccharides. Its presence in the gut is enhanced by diets rich in animal-derived products as found in the typical western diet [David, L. A. et al, Nature (2013) doi:10.1038/nature12820]. Strikingly, prior to treatment, fewer than 0.008% of the 16S-V4 reads from Patient 1 mapped to the genus Bacteroides strongly suggesting that Bacteroides species were absent or that viable Bacteroides were reduced to an extremely minor component of the patient's gut microbiome. Post treatment, ≥42%, of the 16S-V4 reads could be assigned to the genus Bacteroides within 5 days of treatment and by Day 25 post treatment 59.48% of the patients gut microbiome was comprised of Bacteroides. These results were confirmed microbiologically by the absence of detectable Bacteroides in the pretreatment sample plated on two different Bacteroides selective media: Bacteroides Bile Esculin (BBE) agar which is selective for Bacteroides fragilis group species [Livingston, S. J. et al J. Clin. Microbiol (1978). 7: 448-453] and Polyamine Free Arabinose (PFA) agar [Noack et al. J. Nutr. (1998) 128: 1385-1391; modified by replacing glucose with arabinose]. The highly selective BBE agar had a limit of detection of <2×10³ cfu/g, while the limit of detection for Bacteroides on PFA agar was approximately 2×10⁷ cfu/g due to the growth of multiple non-Bacteroides species in the pretreatment sample on that medium. Colony counts of Bacteroides species on Day 25 were up to 2×10¹⁰ cfu/g, consistent with the 16S-V4 sequencing, demonstrating a profound reconstitution of the gut microbiota in Patient 1 (Table 20).

The significant abundance of Bacteroides in Patient 1 on Day 25 (and as early as Day 5 as shown by 16S-V4 sequencing) is remarkable. Viable Bacteroides fragilis group species were not present in the ethanol treated spore population based on microbiological plating (limit of detection of 10 cfu/ml). Thus, administration of the ethanol treated spore population to Patient 1 resulted not only in the engraftment of spore-forming species, but also the restoration of high levels of non-spore forming species commonly found in healthy individuals through the creation of a niche that allowed for the repopulation of Bacteroides species. These organisms were most likely either present at extremely low abundance in the GI tract of Patient 1, or present in a reservoir in the GI tract from which they could rebound to high titer. Those species may also be reinoculated via oral uptake from food following treatment. We term this healthy repopulation of the gut with OTUs that are not present in the ethanol treated spore population “Augmentation.” Augmentation is an important phenomenon in that it shows the ability to use an ethanol treated spore ecology to restore a healthy microbiota by seeding a diverse array or commensal organisms beyond the actual component organisms in the ethanol treated spore population itself; specifically the spore treatment itself and the engraftment of OTUs from the spore composition create a niche that enables the outgrowth of OTUs required to shift a dysbiotic microbiome to a microbial ecology that is associated with health. The diversity of Bacteroides species and their approximate relative abundance in the gut of Patient 1 is shown in Table 21, comprising at least 8 different species.

See FIG. 21: Species Engrafting versus Species Augmenting in patients microbiomes after treatment with an ethanol-treated spore population. Relative abundance of species that engrafted or augmented as described were determined based on the number of 16S sequence reads. Each plot is from a different patient treated with the ethanol-treated spore population for recurrent C. difficile.

The impact of ethanol treated spore population treatment on carriage of imipenem resistant Enterobacteriaceae was assessed by plating pretreatment and Day 28 clinical samples from Patients 2, 4 and 5 on MacConkey lactose plus 1 ug/mL of imipenem. Resistant organisms were scored by morphology, enumerated and DNA was submitted for full length 16S rDNA sequencing as described above. Isolates were identified as Morganella morganii, Providencia rettgeri and Proteus pennerii. Each of these are gut commensal organisms; overgrowth can lead to bacteremia and/or urinary tract infections requiring aggressive antibiotic treatment and, in some cases, hospitalization [Kim, B-N, et al Scan J. Inf Dis (2003) 35: 98-103; Lee, I-K and Liu, J-W J. Microbiol Immunol Infect (2006) 39: 328-334; O'Hara et al, Clin Microbiol Rev (2000) 13: 534]. The titer of organisms at pretreatment and Day 28 by patient is shown in Table 22. Importantly, administration of the ethanol treated spore preparation resulted in greater than 100-fold reduction in 4 of 5 cases of Enterobacteriaceae carriage with multiple imipenem resistant organisms (Table 22).

In addition to speciation and enumeration, multiple isolates of each organism from Patient 4 were grown overnight in 96-well trays containing a 2-fold dilution series of imipenem in order to quantitatively determine the minimum inhibitory concentration (MIC) of antibiotic. Growth of organisms was detected by light scattering at 600 nm on a SpectraMax M5e plate reader. In the clinical setting, these species are considered resistant to imipenem if they have an MIC of 1 ug/mL or greater. M. morganii isolates from pretreatment samples from Patient D had MICs of 2-4 ug/mL and P. pennerii isolates had MICs of 4-8 ug/mL. Thus the ethanol treated spore population administered to Patient 4 caused the clearance of 2 imipenem resistant organisms (Table 16).

Example 26. Enrichment and Purification of Bacteria

To purify individual bacterial strains, dilution plates were selected in which the density enables distinct separation of single colonies. Colonies were picked with a sterile implement (either a sterile loop or toothpick) and re-streaked to BBA or other solid media. Plates were incubated at 37° C. for 3-7 days. One or more well-isolated single colonies of the major morphology type were re-streaked. This process was repeated at least three times until a single, stable colony morphology is observed. The isolated microbe was then cultured anaerobically in liquid media for 24 hours or longer to obtain a pure culture of 10⁶-10¹⁰ cfu/ml. Liquid growth medium might include Brain Heart Infusion-based medium (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with yeast extract, hemin, cysteine, and carbohydrates (for example, maltose, cellobiose, soluble starch) or other media described previously (e.g. see example 14). The culture was centrifuged at 10,000×g for 5 min to pellet the bacteria, the spent culture media was removed, and the bacteria were resuspended in sterile PBS. Sterile 75% glycerol was added to a final concentration of 20%. An aliquot of glycerol stock was titered by serial dilution and plating. The remainder of the stock was frozen on dry ice for 10-15 min and then placed at −80 C for long term storage.

Example 27. Cell Bank Preparation

Cell banks (RCBs) of bacterial strains were prepared as follows. Bacterial strains were struck from −80° C. frozen glycerol stocks to Brucella blood agar with Hem in or Vitamin K (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010), M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) or other solid growth media and incubated for 24 to 48 h at 37° C. in an anaerobic chamber with a gas mixture of H₂:CO₂:N₂ of 10:10:80. Single colonies were then picked and used to inoculate 250 ml to 1 L of Wilkins-Chalgren broth, Brain-Heart Infusion broth, M2GSC broth or other growth media, and grown to mid to late exponential phase or into the stationary phase of growth. Alternatively, the single colonies may be used to inoculate a pilot culture of 10 ml, which were then used to inoculate a large volume culture. The growth media and the growth phase at harvest were selected to enhance cell titer, sporulation (if desired) and phenotypes that might be associated desired in vitro or in vivo. Optionally, Cultures were grown static or shaking, depending which yielded maximal cell titer. The cultures were then concentrated 10 fold or more by centrifugation at 5000 rpm for 20 min, and resuspended in sterile phosphate buffered saline (PBS) plus 15% glycerol. 1 ml aliquots were transferred into 1.8 ml cryovials which were then frozen on dry ice and stored at −80 C. The identity of a given cell bank was confirmed by PCR amplification of the 16S rDNA gene, followed by Sanger direct cycle sequencing, and comparison to a curated rDNA database to determine a taxonomic ID. Each bank was confirmed to yield colonies of a single morphology upon streaking to Brucella blood agar or M2GSC agar. When more than one morphology was observed, colonies were confirmed to be the expected species by PCR and sequencing analysis of the 16S rDNA gene. Variant colony morphologies can be observed within pure cultures, and in a variety of bacteria the mechanisms of varying colony morphologies have been well described (van der Woude, Clinical Microbiology Reviews, 17:518, 2004), including in Clostridium species (Wadsworth-KTL Anaerobic Bacteriology Manual, 6th Ed, Jousimie-Somer, et al 2002). For obligate anaerobes, RCBs were confirmed to lack aerobic colony forming units at a limit of detection of 10 cfu/ml.

Example 28. Titer Determination

The number of viable cells per ml was determined on the freshly harvested, washed and concentrated culture by plating serial dilutions of the RCB to Brucella blood agar or other solid media, and varied from 10⁶ to 10¹⁰ cfu/ml. The impact of freezing on viability was determined by titering the banks after one or two freeze-thaw cycles on dry ice or at −80° C., followed by thawing in an anaerobic chamber at room temperature. Some strains displayed a 1-3 log drop in viable cfu/ml after the 1st and/or 2nd freeze thaw, while the viability of others were unaffected.

Example 29. Preparation of Bacterial Compositions

Individual strains were typically thawed on ice and combined in an anaerobic chamber to create mixtures, followed by a second freeze at −80° C. to preserve the mixed samples. When making combinations of strains for in vitro or in vivo assays, the cfu in the final mixture was estimated based on the second freeze-thaw titer of the individual strains. For experiments in rodents, strains may be combined at equal counts in order to deliver between 1e4 and 1e10 per strain. Additionally, some bacteria may not grow to sufficient titer to yield cell banks that allowed the production of compositions where all bacteria were present at 1e10.

Example 30. Identification of Keystone OTUs and Functions

The human body is an ecosystem in which the microbiota, and the microbiome, play a significant role in the basic healthy function of human systems (e.g. metabolic, immunological, and neurological). The microbiota and resulting microbiome comprise an ecology of microorganisms that co-exist within single subjects interacting with one another and their host (i.e., the mammalian subject) to form a dynamic unit with inherent biodiversity and functional characteristics. Within these networks of interacting microbes (i.e. ecologies), particular members can contribute more significantly than others; as such these members are also found in many different ecologies, and the loss of these microbes from the ecology can have a significant impact on the functional capabilities of the specific ecology. Robert Paine coined the concept “Keystone Species” in 1969 (see Paine R T. 1969. A note on trophic complexity and community stability. The American Naturalist 103: 91-93.) to describe the existence of such lynchpin species that are integral to a given ecosystem regardless of their abundance in the ecological community. Paine originally describe the role of the starfish Pisaster ochraceus in marine systems and since the concept has been experimentally validated in numerous ecosystems.

Keystone OTUs and/or Functions are computationally-derived by analysis of network ecologies elucidated from a defined set of samples that share a specific phenotype. Keystone OTUs and/or Functions are defined as all Nodes within a defined set of networks that meet two or more of the following criteria. Using Criterion 1, the node is frequently observed in networks, and the networks in which the node is observed are found in a large number of individual subjects; the frequency of occurrence of these Nodes in networks and the pervasiveness of the networks in individuals indicates these Nodes perform an important biological function in many individuals. Using Criterion 2, the node is frequently observed in networks, and each the networks in which the node is observed contain a large number of Nodes—these Nodes are thus “super-connectors”, meaning that they form a nucleus of a majority of networks and as such have high biological significance with respect to their functional contributions to a given ecology. Using Criterion 3, the node is found in networks containing a large number of Nodes (i.e. they are large networks), and the networks in which the node is found occur in a large number of subjects; these networks are potentially of high interest as it is unlikely that large networks occurring in many individuals would occur by chance alone strongly suggesting biological relevance. Optionally, the required thresholds for the frequency at which a node is observed in network ecologies, the frequency at which a given network is observed across subject samples, and the size of a given network to be considered a Keystone node are defined by the 50th, 70th, 80th, or 90th percentiles of the distribution of these variables. Optionally, the required thresholds are defined by the value for a given variable that is significantly different from the mean or median value for a given variable using standard parametric or non-parametric measures of statistical significance. In another embodiment a Keystone node is defined as one that occurs in a sample phenotype of interest such as but not limited to “health” and simultaneously does not occur in a sample phenotype that is not of interest such as but not limited to “disease.” Optionally, a Keystone Node is defined as one that is shown to be significantly different from what is observed using permuted test datasets to measure significance.

Example 31. Identifying the Core Ecology from the Ethanol Treated Spore Preparation

Ten different ethanol treated spore preparations were made from 6 different donors (as described in Example 15). The spore preparations were used to treat 10 patients, each suffering from recurrent C. difficile infection. Patients were identified using the inclusion/exclusion criteria described in Example 18, and donors were identified using the criteria described in Example 1. None of the patients experienced a relapse of C. difficile in the 4 weeks of follow up after treatment, whereas the literature would predict that 70-80% of subjects would experience a relapse following cessation of antibiotic [Van Nood, et al, NEJM (2013)]. Thus, the ethanol treated spore preparations derived from multiple different donors and donations showed remarkable clinical efficacy.

To define the Core Ecology underlying the remarkable clinical efficacy of the ethanol treated spore preparation, the following analysis was carried out. The OTU composition of the spore preparation was determined by 16S-V4 rDNA sequencing and computational assignment of OTUs per Example 12. A requirement to detect at least ten sequence reads in the ethanol treated spore preparation was set as a conservative threshold to define only OTUs that were highly unlikely to arise from errors during amplification or sequencing. Methods routinely employed by those familiar to the art of genomic-based microbiome characterization use a read relative abundance threshold of 0.005% (see e.g. Bokulich, A. et al. 2013. Quality-filtering vastly improves diversity estimates from IIlumina amplicon sequencing. Nature Methods 10: 57-59), which would equate to ≥2 reads given the sequencing depth obtained for the samples analyzed in this example, as cut-off which is substantially lower than the ≥10 reads used in this analysis. All taxonomic and clade assignments were made for each OTU as described in Examples 12. The resulting list of OTUs, clade assignments, and frequency of detection in the spore preparations are shown in Table GB. OTUs that engraft in a treated patients and the percentage of patients in which they engraft are denoted, as are the clades, spore forming status, and Keystone OTU status. Starred OTUs occur in ≥80% of the ethanol preps and engraft in ≥50% of the treated patients.

TABLE GB OTUs detected by a minimum of ten 16S-V4 sequence reads in at least a one ethanol treated spore preparation (pan-microbiome). % of % of Spore Patients Preps OTU Spore Keystone OTU Clade with OTU Engrafts Former OTU Prevotella_maculosa clade_104 10%  0% N N Prevotella_copri clade_168 20%  0% N N Bacteroides_caccae clade_170 30%  0% N Y Bifidobacterium_sp_TM_7* clade_172 90% 60% N N Bifidobacterium_gallicum clade_172 70% 20% N N Bifidobacterium_dentium clade_172 50%  0% N N Lactobacillus_casei clade_198 20% 10% N N Actinomyces_odontolyticus clade_212 20% 30% N N Clostridium_colicanis clade_223 10% 10% Y N Clostridiales_sp_SS3_4* clade_246 100%  70% Y N Clostridium_sporogenes clade_252 40% 40% Y N Clostridium_butyricum clade_252 20% 20% Y N Clostridium_disporicum clade_253 40% 30% Y N Clostridium_hylemonae* clade_260 100%  50% Y N Clostridium_scindens clade_260 10% 60% Y N Coprococcus_comes* clade_262 90% 80% Y Y Lachnospiraceae_bacterium_1_4_56FAA* clade_262 90% 80% Y Y Ruminococcus_torques clade_262 30% 70% Y Y Parabacteroides_merdae clade_286 30% 20% N Y Bifidobacterium_bifidum clade_293 10%  0% N N Johnsonella_ignava clade_298 10% 10% N N Blautia_glucerasea* clade_309 100%  80% Y N Blautia_sp_M25* clade_309 100%  70% Y Y Lachnospiraceae_bacterium_6_1_63FAA* clade_309 100%  60% Y N Eubacterium_cellulosolvens clade_309 10% 30% Y Y Lactobacillus_fermentum clade_313 10%  0% N N Sarcina_ventriculi clade_353 10% 10% Y N Clostridium_bartlettii* clade_354 90% 70% Y N Clostridium_bifermentans clade_354 70% 70% Y N Clostridium_mayombei clade_354 50% 50% Y N Dorea_longicatena* clade_360 100%  60% Y Y Lachnospiraceae_bacterium_9_1_43BFAA clade_360 100%  30% Y N Lachnospiraceae_bacterium_2_1_58FAA* clade_360 80% 80% Y N Lachnospiraceae_bacterium_2_1_46FAA clade_360 50% 50% Y N Lactobacillus_perolens clade_373 10%  0% N N Bacteroides_dorei clade_378 60% 50% N Y Eubacterium_biforme clade_385 10%  0% Y N Peptoniphilus_sp_gpac077 clade_389 10% 20% N N Coprococcus_catus* clade_393 100%  70% Y Y Eubacterium_hallii* clade_396 90% 60% Y Y Anaerosporobacter_mobilis clade_396 40% 60% Y N Bacteroides_pectinophilus clade_396 10% 60% Y N Lactobacillus_hominis clade_398 10%  0% N N Lactococcus_lactis clade_401 40% 40% N N Ruminococcus_champanellensis* clade_406 80% 50% Y N Ruminococcus_callidus clade_406 10% 10% Y N Clostridium_clostridioforme* clade_408 100%  60% Y Y Eubacterium_hadrum* clade_408 100%  90% Y Y Clostridium_symbiosum clade_408 30% 50% Y Y Anaerostipes_caccae clade_408 10% 50% Y N Parasutterella_excrementihominis clade_432 10%  0% N N Sutterella_stercoricanis clade_432 10%  0% N N Eubacterium_rectale* clade_444 100%  80% Y Y Lachnobacterium_bovis* clade_444 100%  80% Y N Desulfovibrio_desulfuricans clade_445 10%  0% N Y Eubacterium_sp_oral_clone_JS001* clade_476 80% 70% Y N Faecalibacterium_prausnitzii* clade_478 100%  60% Y Y Subdoligranulum_variabile* clade_478 100%  80% Y Y Coprobacillus_sp_D7* clade_481 90% 60% Y N Clostridium_cocleatum clade_481 60% 20% Y N Clostridium_spiroforme clade_481 40% 50% Y N Eubacterium_ramulus* clade_482 80% 60% Y N Flavonifractor_plautii clade_494 70% 60% Y Y Pseudoflavonifractor_capillosus clade_494 60% 60% Y Y Ruminococcaceae_bacterium_D16 clade_494 30% 50% Y Y Acetivibrio_cellulolyticus* clade_495 70% 80% Y N Clostridium_stercorarium clade_495 40% 50% Y N Enterococcus_durans clade_497 10% 10% N N Enterococcus_faecium clade_497 10% 10% N N Dialister_invisus clade_506 50% 10% N N Eubacterium_limosum clade_512 20%  0% Y N Ruminococcus_flavefaciens clade_516 60% 60% Y N Eubacterium_ventriosum clade_519 30% 60% Y Y Bilophila_wadsworthia clade_521 90%  0% N Y Lachnospira_pectinoschiza clade_522 40% 60% Y N Eubacterium_eligens clade_522 30% 50% Y Y Catonella_morbi clade_534 20%  0% N N Clostridium_sporosphaeroides* clade_537 100%  80% Y N Ruminococcus_bromii clade_537 60% 30% Y Y Clostridium_leptum clade_537 40% 70% Y Y Clostridium_sp_YIT_12069 clade_537 40% 60% Y N Clostridium_viride clade_540 10% 10% Y N Megamonas_funiformis clade_542 50%  0% N N Eubacterium_ruminantium* clade_543 80% 90% Y N Coprococcus_eutactus clade_543 20% 20% Y N Collinsella_aerofaciens clade_553 50% 10% Y Y Alkaliphilus_metalliredigenes clade_554 40% 10% Y N Turicibacter_sanguinis clade_555 80% 40% Y N Phascolarctobacterium_faecium clade_556 20%  0% N N Clostridiales_bacterium_oral_clone_P4PA* clade_558 80% 50% N N Lutispora_thermophila clade_564 100%   0% Y N Coriobacteriaceae_bacterium_JC110 clade_566 70%  0% N N Eggerthella_sp_1_3_56FAA clade_566 70% 30% N N Adlercreutzia_equolifaciens clade_566 40%  0% N N Gordonibacter_pamelaeae clade_566 30%  0% N Y Slackia_isoflavoniconvertens clade_566 10%  0% N N Eubacterium_desmolans* clade_572 90% 70% Y N Papillibacter_cinnamivorans* clade_572 90% 80% Y N Clostridium_colinum clade_576 30% 30% Y N Akkermansia_muciniphila clade_583 60% 10% N Y Clostridiales_bacterium_oral_taxon_F32 clade_584 60% 30% N N Prochlorococcus_marinus clade_592 30%  0% N N Methanobrevibacter_wolinii clade_595 30%  0% N N Bacteroides_fragilis clade_65 20% 30% N Y Lactobacillus_delbrueckii clade_72 10%  0% N N Escherichia_coli clade_92 50%  0% N Y Clostridium_sp_D5 clade_96 80% 60% Y N Streptococcus_thermophilus clade_98 90% 20% N Y Streptococcus_sp_CM6 clade_98 20% 10% N N Streptococcus_sp_oral_clone_ASCE05 clade_98 10%  0% N N

Next, it was reasoned that for an OTU to be considered a member of the Core Ecology of the spore preparation, that OTU must be shown to engraft in a patient. Engraftment is important for two reasons. First, engraftment is a sine qua non of the mechanism to reshape the microbiome and eliminate C. difficile colonization. OTUs that engraft with higher frequency are highly likely to be a component of the Core Ecology of the spore preparation. Second, OTUs detected by sequencing the spore preparation (as in Table GB) may include non-viable spores or other contaminant DNA molecules not associated with spores. The requirement that an OTU must be shown to engraft in the patient eliminates OTUs that represent non-viable spores or contaminating sequences. Table GB also identifies all OTUs detected in the spore preparation that also were shown to engraft in at least one patient post-treatment. OTUs that are present in a large percentage of the ethanol spore preparations analyzed and that engraft in a large number of patients represent a subset of the Core Ecology that are highly likely to catalyze the shift from a dysbiotic disease ecology to a healthy microbiome.

A third lens was applied to further refine insights into the Core Ecology of the spore preparation. Computational-based, network analysis has enabled the description of microbial ecologies that are present in the microbiota of a broad population of healthy individuals. These network ecologies are comprised of multiple OTUs, some of which are defined as Keystone OTUs. Keystone OTUs are computationally defined as described in Example 30. Keystone OTUs form a foundation to the microbially ecologies in that they are found and as such are central to the function of network ecologies in healthy subjects. Keystone OTUs associated with microbial ecologies associated with healthy subjects are often are missing or exist at reduced levels in subjects with disease. Keystone OTUs may exist in low, moderate, or high abundance in subjects. Table GB further notes which of the OTUs in the spore preparation are Keystone OTUs exclusively associated with individuals that are healthy and do not harbor disease.

There are several important findings from this data. A relatively small number of species, 16 in total, are detected in all of the spore preparations from 6 donors and 10 donations. This is surprising because the HMP database (www.hmpdacc.org) describes the enormous variability of commensal species across healthy individuals. The presence of a small number of consistent OTUs lends support to the concept of a Core Ecology. The engraftment data further supports this conclusion. A regression analysis shows a significant correlation between frequency of detection in a spore preparation and frequency of engraftment in a donor: R=0.43 (p<0.001). There is no a priori requirement that an OTU detected frequently in the spore preparation will or should engraft. For instance, Lutispora thermophila, a spore former found in all ten spore preparations, did not engraft in any of the patients. Bilophila wadsworthia, a gram negative anaerobe, is present in 9 of 10 donations, yet it does not engraft in any patient, indicating that it is likely a non-viable contaminant in the ethanol treated spore preparation. Finally, it is worth noting the high preponderance of previously defined Keystone OTUs among the most frequent OTUs in the spore preparations.

These three factors—prevalence in the spore preparation, frequency of engraftment, and designation as a Keystone OTUs—enabled the creation of a “Core Ecology Score” (CES) to rank individual OTUs. CES was defined as follows:

-   -   40% weighting for presence of OTU in spore preparation     -   multiplier of 1 for presence in 1-3 spore preparations     -   multiplier of 2.5 for presence in 4-8 spore preparations     -   multiplier of 5 for presences in 9 spore preparations     -   40% weighting for engraftment in a patient     -   multiplier of 1 for engraftment in 1-4 patients     -   multiplier of 2.5 for engraftment in 5-6 patients     -   multiplier of 5 for engraftment in 7 patients     -   20% weighting to Keystone OTUs     -   multiplier of 1 for a Keystone OTU     -   multiplier of 0 for a non-Keystone OTU

Using this guide, the CES has a maximum possible score of 5 and a minimum possible score of 0.8. As an example, an OTU found in 8 of the 10 spore preparations that engrafted in 3 patients and was a Keystone OTU would be assigned the follow CES:

CES=(0.4×2.5)+(0.4×1)+(0.2×1)=1.6

Table GC ranks the top 20 OTUs by CES with the further requirement that an OTU must be shown to engraft to be a considered an element of a core ecology.

TABLE GC Top 20 OTUs ranked by CES Spore Keystone OTU Clade CES Former OTU Eubacterium_hadrum clade_408 4.2 Y Y Eubacterium_rectale clade_444 4.2 Y Y Subdoligranulum_variabile clade_478 4.2 Y Y Blautia_sp_M25 clade_309 4.2 Y Y Coprococcus_catus clade_393 4.2 Y Y Lachnospiraceae_bacterium_1_4_56FAA clade_262 4.2 Y Y Coprococcus_comes clade_262 4.2 Y Y Blautia_glucerasea clade_309 4.0 Y N Lachnobacterium_bovis clade_444 4.0 Y N Clostridium_sporosphaeroides clade_537 4.0 Y N Clostridiales_sp_SS3_4 clade_246 4.0 Y N Papillibacter_cinnamivorans clade_572 4.0 Y N Clostridium_bartlettii clade_354 4.0 Y N Eubacterium_desmolans clade_572 4.0 Y N Clostridium_clostridioforme clade_408 3.2 Y Y Dorea_longicatena clade_360 3.2 Y Y Faecalibacterium_prausnitzii clade_478 3.2 Y Y Eubacterium_hallii clade_396 3.2 Y Y Clostridium_leptum clade_537 3.2 Y Y Lachnospiraceae_bacterium_6_1_63FAA clade_309 3.0 Y N

Example 32. Defining Efficacious Subsets of the Core Ecology

The number of organisms in the human gastrointestinal tract, as well as the diversity between healthy individuals, is indicative of the functional redundancy of a healthy gut microbiome ecology (see The Human Microbiome Consortia. 2012. Structure, function and diversity of the healthy human microbiome. Nature 486: 207-214). This redundancy makes it highly likely that subsets of the Core Ecology describe therapeutically beneficial components of the ethanol treated spore preparation and that such subsets may themselves be useful compositions for the treatment of C. difficile infection given the ecologies functional characteristics. Using the CES, individual OTUs can be prioritized for evaluation as an efficacious subset of the Core Ecology.

Another aspect of functional redundancy is that evolutionarily related organisms (i.e. those close to one another on the phylogenetic tree, e.g. those grouped into a single clade) will also be effective substitutes in the Core Ecology or a subset thereof for treating C. difficile.

To one skilled in the art, the selection of appropriate OTU subsets for testing in vitro (e.g. see Example 33 below) or in vivo (e.g. see Examples 16 or 17) is straightforward. Subsets may be selected by picking any 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 OTUs from Table GB, with a particular emphasis on those with higher CES, such as the OTUs described in Table GC. In addition, using the clade relationships defined in Example 12 and Table 1 above, related OTUs can be selected as substitutes for OTUs with acceptable CES values. These organisms can be cultured anaerobically in vitro using the appropriate media (selected from those described in Example 14 above), and then combined in a desired ratio. A typical experiment in the mouse C. difficile model utilizes at least 10⁴ and preferably at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or more than 10⁹ colony forming units of a each microbe in the composition. Variations in the culture yields may sometimes mean that organisms are combined in unequal ratios, e.g. 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, or greater than 1:100,000. What is important in these compositions is that each strain be provided in a minimum amount so that the strain's contribution to the efficacy of the Core Ecology subset can be measured. Using the principles and instructions described here, it is straightforward for one of skill in the art to make clade-based substitutions to test the efficacy of subsets of the Core Ecology. Table GB describes the clades for each OTU detected in a spore preparation and Table 1 describes the OTUs that can be used for substitutions based on clade relationships.

Example 33. Testing Subsets of the Core Ecology in the Mouse Model

Several subsets of the Core Ecology were tested in the C. difficile mouse model. The negative control was phosphate buffered saline and the positive control was a 10% human fecal suspension. The subsets are described in Table GD.

TABLE GD Subsets of the Core Ecology tested in the C. difficile mouse model Subset OTU Substitute For OTU in Table 1 (Clade) Subset 1 Collinsella aerofaciens none (Clade_553) Clostridium tertium C. sporogenes (Clade_252) Clostridium disporicum none (Clade_253) Clostridium innocuum Clostridium_sp_HGF2 (Clade_351) Clostridium mayombei none (Clade_354) Clostridium butyricum none (Clade_252) Coprococcus comes none (Clade_262) Clostridium hylemonae none (Clade_260) Clostridium bolteae E. hadrum (Clade_408) Clostridium symbiosum C. clostridioforme (Clade_408) Clostridium orbiscindens R._bacterium_D16 (Clade_494) Lachnospiraceae bacterium_5_1_57FAA C. scindens (Clade_260) Blautia producta Blautia_sp_M25 (Clade_309) Ruminococcus gnavus D. longicatena(Clade_360) Ruminococcus bromii none (Clade_537) Subset 2 Collinsella aerofaciens none (Clade_553) Clostridium butyricum none (Clade_252) Clostridium hylemonae none (Clade_260) Blautia producta Blautia_sp_M25 (Clade_309) Subset 3 Collinsella aerofaciens none (Clade_553) Clostridium innocuum Clostridium_sp_HGF2 (Clade_351) Coprococcus comes none (Clade_262) Ruminococcus bromii none (Clade_537) Subset 4 Clostridium butyricum none (Clade_252) Clostridium hylemonae none (Clade_260) Blautia producta Blautia_sp_M25 (Clade_309) Subset 5 Clostridium butyricum none (Clade_252) Clostridium hylemonae none (Clade_260) Subset 6 Blautia producta Blautia_sp_M25 (Clade_309) Clostridium butyricum none (Clade_252) Subset 7 Clostridium orbiscindens R._bacterium_D16 (Clade_494) Lachnospiraceae bacterium_5_1_57FAA C. scindens (Clade_260) Eubacterium rectale none (Clade_444)

Two cages of five mice each were tested for each arm of the experiment. All mice received an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and Vancomycin (0.056 mg/ml) in their drinking water on days −14 through −5 and a dose of 10 mg/kg Clindamycin by oral gavage on day −3. On day −1, they received either the test articles or control articles via oral gavage. On day 0 they were challenged by administration of approximately 4.5 log 10 cfu of C. difficile (ATCC 43255) via oral gavage. Mortality was assessed every day from day 0 to day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the empty vehicle was used to assess the success of the test article. Additionally, a C. difficile symptom scoring was performed each day from day −1 through day 6. Symptom scoring was based on Appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), Respiration (0-2 pts based on normal, rapid or shallow, with abdominal breathing), Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).

In addition to compiling the cumulative mortality for each arm, the average minimum relative weight is calculated as the mean of each mouse's minimum weight relative to Day −1 and the average maximum clinical score is calculated as the mean of each mouse's maximum combined clinical score with a score of 4 assigned in the case of death. The results are reported in Table GE.

TABLE GE Results of bacterial compositions tested in a C. difficile mouse model. Cumulative Avg. Maximum Mortality Avg. Minimum Clinical Score Group Dose (%) Relative Weight (Death = 4) Vehicle Control — 40 0.87 2.8 Feces Control 5.8e8 cfu total 0 0.99 0 Subset 1 1e8 cfu/OTU 0 0.98 0 Subset 2 1e8 cfu/OTU 10 0.84 2.1 Subset 3 1e8 cfu/OTU 10 0.84 2.2 Subset 4 1e8 cfu/OTU 0 0.87 2 Subset 5 1e8 cfu/OTU 20 0.91 1.7 Subset 6 1e8 cfu/OTU 40 0.82 2.8 Subset 7 1e8 cfu/OTU 0 0.90 1

Example 34. Defining Subsets of the Core Ecology in the In Vitro C. difficile Inhibition Assay

Vials of −80° C. glycerol stock banks were thawed and diluted to 1e8 CFU/mL. Selected strains and their clade assignment are given in Table GF. Each strain was then diluted 10× (to a final concentration of 1e7 CFU/mL of each strain) into 200 uL of PBS+15% glycerol in the wells of a 96-well plate. Plates were then frozen at −80° C. When needed for the assay, plates were removed from −80° C. and thawed at room temperature under anaerobic conditions when testing in a in vitro C. difficile inhibition assay (CivSim).

An overnight culture of Clostridium difficile is grown under anaerobic conditions in SweetB-FosIn or other suitable media for the growth of C. difficile. SweetB-FosIn is a complex media composed of brain heart infusion, yeast extract, cysteine, cellobiose, maltose, soluble starch, and fructooligosaccharides/inulin, and hemin, and is buffered with MOPs. After 24 hr of growth the culture is diluted 100,000 fold into a complex media such as SweetB-FosIn which is suitable for the growth of a wide variety of anaerobic bacterial species. The diluted C. difficile mixture is then aliquoted to wells of a 96-well plate (180 uL to each well). 20 uL of a subset Core Ecology is then added to each well at a final concentration of 1e6 CFU/mL of each species. Alternatively the assay can be tested each species at different initial concentrations (1e9 CFU/mL, 1e8 CFU/mL, 1e7 CFU/mL, 1e5 CFU/mL, 1e4 CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL). Control wells only inoculated with C. difficile are included for a comparison to the growth of C. difficile without inhibition. Additional wells are used for controls that either inhibit or do not inhibit the growth of C. difficile. One example of a positive control that inhibits growth is a combination of Blautia producta, Clostridium bifermentans and Escherichia coli. One example of a control that shows reduced inhibition of C. difficile growth is a combination of Bacteroides thetaiotaomicron, Bacteroides ovatus and Bacteroides vulgatus. Plates are wrapped with parafilm and incubated for 24 hr at 37° C. under anaerobic conditions. After 24 hr the wells containing C. difficile alone are serially diluted and plated to determine titer. The 96-well plate is then frozen at −80 C before quantifying C. difficile by qPCR assay.

A standard curve is generated from a well on each assay plate containing only pathogenic C. difficile grown in SweetB+FosIn media and quantified by selective spot plating. Serial dilutions of the culture are performed in sterile phosphate-buffered saline. Genomic DNA is extracted from the standard curve samples along with the other wells.

Genomic DNA is extracted from 5 μl of each sample using a dilution, freeze/thaw, and heat lysis protocol. 5 μL of thawed samples is added to 45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) and mixed by pipetting. The plates with diluted samples are frozen at −20° C. until use for qPCR which includes a heated lysis step prior to amplification. Alternatively the genomic DNA is isolated using the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

The qPCR reaction mixture contains 1× SsoAdvanced Universal Probes Supermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG (SEQ ID NO: 2040), IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer (CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 2041), IDT, Coralville, Iowa), 250 nM of Wr-tcdB-P probe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 2042), Life Technologies, Grand Island, N.Y.), and Molecular Biology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl (Primers adapted from: Wroblewski, D. et al., Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens, Journal of Clinical Microbiology 47:2142-2148 (2009)). This reaction mixture is aliquoted to wells of a Hard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate (BioRad, Hercules, Calif.). To this reaction mixture, 2 μl of diluted, frozen, and thawed samples are added and the plate sealed with a Microseal ‘B’ Adhesive Seal (BioRad, Hercules, Calif.). The qPCR is performed on a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System (BioRad, Hercules, Calif.). The thermocycling conditions are 95° C. for 15 minutes followed by 45 cycles of 95° C. for 5 seconds, 60° C. for 30 seconds, and fluorescent readings of the FAM channel. Alternatively, the qPCR is performed with other standard methods known to those skilled in the art.

The Cq value for each well on the FAM channel is determined by the CFX Manager™ 3.0 software. The log₁₀(cfu/mL) of C. difficile each experimental sample is calculated by inputting a given sample's Cq value into a linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log₁₀(cfu/mL) of those samples. The log inhibition is calculated for each sample by subtracting the log₁₀(cfu/mL) of C. difficile in the sample from the log₁₀(cfu/mL) of C. difficile in the sample on each assay plate used for the generation of the standard curve that has no additional bacteria added. The mean log inhibition is calculated for all replicates for each composition.

A histogram of the range and standard deviation of each composition is plotted. Ranges or standard deviations of the log inhibitions that are distinct from the overall distribution are examined as possible outliers. If the removal of a single log inhibition datum from one of the binary pairs that is identified in the histograms would bring the range or standard deviation in line with those from the majority of the samples, that datum is removed as an outlier, and the mean log inhibition is recalculated.

The pooled variance of all samples evaluated in the assay is estimated as the average of the sample variances weighted by the sample's degrees of freedom. The pooled standard error is then calculated as the square root of the pooled variance divided by the square root of the number of samples. Confidence intervals for the null hypothesis are determined by multiplying the pooled standard error to the z score corresponding to a given percentage threshold. Mean log inhibitions outside the confidence interval are considered to be inhibitory if positive or stimulatory if negative with the percent confidence corresponding to the interval used. Ternary combinations with mean log inhibition greater than 0.312 are reported as ++++ (≥99% confidence interval (C.I.) of the null hypothesis), those with mean log inhibition between 0.221 and 0.312 as +++ (95%<C.I.<99%), those with mean log inhibition between 0.171 and 0.221 as ++ (90%<C.I.<95%), those with mean log inhibition between 0.113 and 0.171 as + (80%<C.I.<90%), those with mean log inhibition between −0.113 and −0.171 as − (80%<C.I.<90%), those with mean log inhibition between −0.171 and −0.221 as −− (90%<C.I.<95%), those with mean log inhibition between −0.221 and −0.312 as −−− (95%<C.I.<99%), and those with mean log inhibition less than −0.312 as −−−− (99%<C.I.).

TABLE GF OTUs and their clade assignments tested in ternary combinations with results in the in vitro inhibition assay OTU1 Clade1 OTU2 Clade2 OTU3 Clade3 Results Clostridium_bolteae clade_408 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++ Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 Blautia_producta clade_309 ++++ Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 Eubacterium_rectale clade_444 − Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 − Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Clostridium_bolteae clade_408 Faecalibacterium_prausnitzii clade_478 Blautia_producta clade_309 ++++ Clostridium_bolteae clade_408 Faecalibacterium_prausnitzii clade_478 Eubacterium_rectale clade_444 Clostridium_bolteae clade_408 Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 ++++ Clostridium_bolteae clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 ++++ Clostridium_bolteae clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Eubacterium_rectale clade_444 + Clostridium_symbiosum clade_408 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++ Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 Blautia_producta clade_309 ++++ Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 Eubacterium_rectale clade_444 Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 + Clostridium_symbiosum clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 ++++ Clostridium_symbiosum clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Eubacterium_rectale clade_444 Collinsella_aerofaciens clade_553 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++ Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408 Blautia_producta clade_309 ++++ Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 ++++ Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408 Eubacterium_rectale clade_444 ++++ Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408 Faecalibacterium_prausnitzii clade_478 ++++ Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 ++++ Collinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408 Blautia_producta clade_309 ++++ Collinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408 Eubacterium_rectale clade_444 Collinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 Collinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 + Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Blautia_producta clade_309 ++++ Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Clostridium_bolteae clade_408 ++++ Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 +++ Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Eubacterium_rectale clade_444 +++ Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Faecalibacterium_prausnitzii clade_478 ++++ Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Lachnospiraceae_bacterium_5_1_57FAA clade_260 +++ Collinsella_aerofaciens clade_553 Faecalibacterium_prausnitzii clade_478 Blautia_producta clade_309 ++++ Collinsella_aerofaciens clade_553 Faecalibacterium_prausnitzii clade_478 Eubacterium_rectale clade_444 +++ Collinsella_aerofaciens clade_553 Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 +++ Collinsella_aerofaciens clade_553 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 ++++ Collinsella_aerofaciens clade_553 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Eubacterium_rectale clade_444 ++++ Coprococcus_comes clade_262 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++ Coprococcus_comes clade_262 Clostridium_bolteae clade_408 Blautia_producta clade_309 ++++ Coprococcus_comes clade_262 Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408 Coprococcus_comes clade_262 Clostridium_bolteae clade_408 Eubacterium_rectale clade_444 −− Coprococcus_comes clade_262 Clostridium_bolteae clade_408 Faecalibacterium_prausnitzii clade_478 +++ Coprococcus_comes clade_262 Clostridium_bolteae clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 +++ Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 Blautia_producta clade_309 ++++ Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 Eubacterium_rectale clade_444 −−− Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 Faecalibacterium_prausnitzii clade_478 Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Coprococcus_comes clade_262 Faecalibacterium_prausnitzii clade_478 Blautia_producta clade_309 ++++ Coprococcus_comes clade_262 Faecalibacterium_prausnitzii clade_478 Eubacterium_rectale clade_444 − Coprococcus_comes clade_262 Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Coprococcus_comes clade_262 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 ++++ Coprococcus_comes clade_262 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Eubacterium_rectale clade_444 Faecalibacterium_prausnitzii clade_478 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++ Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 ++++ Faecalibacterium_prausnitzii clade_478 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Eubacterium_rectale clade_444 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++

The CivSim shows that many ternary combinations inhibit C. difficile. 39 of 56 combinations show inhibition with a confidence interval >80%; 36 of 56 with a C.I.>90%; 36 of 56 with a C.I.>95%; 29 of 56 with a C.I. of >99%. Non-limiting but exemplary ternary combinations include those with mean log reduction greater than 0.171, e.g. any combination shown in Table 6 with a score of ++++, such as Colinsella aerofaciens, Coprococcus comes, and Blautia producta. Equally important, the CivSim assay describes ternary combinations that do not effectively inhibit C. difficile. 5 of 56 combinations promote growth with >80% confidence; 2 of 56 promote growth with >90% confidence; 1 of 56, Coprococcus comes, Clostridium symbiosum and Eubacterium rectale, promote growth with >95% confidence. 12 of 56 combinations are neutral in the assay, meaning they neither promote nor inhibit C. difficile growth to the limit of measurement.

It is straightforward for one of skill in the art to use the CivSim method to determine efficacious subsets of the Core Ecology derived from the ethanol treated spore fraction shown to be efficacious in treating C. difficile in humans.

Example AAZA: Bacterial Compositions Populating the Gut in a Mouse Model

Two bacterial compositions were evaluated in a mouse model to demonstrate the ability to populate the gastrointestinal tract. Bacteria were grown as described in ***Example 14. Compositions were pre-made under anaerobic conditions and suspended in PBS+15% glycerol and stored at ≥−70° C. prior to

Groups of mice (10 females/group; 5 per cage) were pre-treated on Days −14 to −5 with an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and vancomycin (0.056 mg/ml) in their drinking water. On Day −3 they received 10 mg/kg Clindamycin by oral gavage. On Day −1, they were dosed with a microbial compositions by oral gavage in a volume of 0.2 mL (Table ZA). Microbial compositions comprised approximately equal numbers of each OTU and were dosed at approximately 1×10⁹, 1×10⁸ and 1×10⁷ per OTU for each composition (e.g. microbial composition 1, comprising 15 strains, was dosed at approximately 1.5×10¹⁰, 1.5×10⁹, and 1.5×10⁸ total CFU). Fecal samples were collected from each cage on Day −1 (approximately 1 hour before dosing) and on Days 2, 3 and 4 post-dosing. Feces were stored frozen prior to processing and sequencing. Weight gain of mice treated with either microbial compositions was similar to that of naive, control mice.

In parallel, groups of animals treated with the same microbial compositions on Day −1 were challenged on Day 0 with approximately 10⁴⁵ spores of Clostridium difficile (ATCC 43255) via oral gavage. Mortality for C. difficile challenged animals was assessed every day from Day 0 to Day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the empty vehicle was used to assess the success of the test article.

TABLE ZA Microbial compositions administered via oral gavage on Day −1 OTU Clade Microbial Composition 1 Clostridium_butyricum clade_252 Clostridium_disporicum clade_253 Clostridium_hylemonae clade_260 Clostridium_orbiscindens clade_494 Clostridium_symbiosum clade_408 Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Ruminococcus_bromii clade_537 Blautia_producta clade_309 Clostridium_bolteae clade_408 Clostridium_innocuum clade_351 Clostridium_mayombei clade_354 Clostridium_tertium clade_252 Ruminococcus_gnavus clade_360 Microbial Composition 2 Clostridium_disporicum clade_253 Clostridium_orbiscindens clade_494 Clostridium_symbiosum clade_408 Collinsella_aerofaciens clade_553 Eubacterium_rectale clade_444 Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309 Clostridium_innocuum clade_351 Clostridium_mayombei clade_354

Fecal samples were processed by isolating and sequencing DNA according to ***Example 11 and 12. The OTU assignment of fecal samples from Days −1, 2, 3 and 4 was determined by analyzing 16S-V4 sequence reads and assigning OTUs as described in ***Example 11. Clades were assigned as described in ***Example 11. Total read counts were determined for each OTU or each clade by summing the results from cages of the same experimental group. Samples with 10 or fewer sequence reads for a given OTU or clade were considered to be below background and were not included in the summation process. Results are shown by OTU (Table TAB) and by clade (Table TAC).

TABLE TAB Population of OTUs on Days 2, 3 and 4 following dosing with Microbial Compositions 1 × 10⁹ per OTU 1 × 10⁸ per OTU 1 × 10⁷ per OTU −1 2 3 4 −1 2 3 4 −1 2 3 4 Microbial comp 1 Cl_butyricum 0 106 51 32 0 10 0 34 195 0 0 0 Cl_disporicum 10 1746 1190 887 0 1746 769 1011 201 11175 1531 1152 Cl_hylemonae 0 258 258 84 0 203 164 77 0 265 214 90 Cl_orbiscindens 0 188 192 471 0 188 138 276 0 221 174 341 Cl_symbiosum 0 485 482 486 0 444 379 447 0 562 427 775 Co_aerofaciens 0 0 0 0 0 0 0 0 0 0 0 0 C_comes 0 0 0 0 0 0 0 0 0 0 0 0 L_bacterium_5_1_57FAA 0 341 336 354 0 351 182 356 0 256 240 300 R_bromii 0 0 0 0 0 0 0 0 0 0 0 0 B_producta 0 0 0 0 0 0 0 0 0 0 0 0 Cl_bolteae 0 0 0 0 0 0 0 0 0 0 0 0 Cl_innocuum 0 0 0 0 0 0 0 0 0 0 0 0 Cl_mayombei 0 0 0 0 0 0 0 0 0 0 0 0 Cl_tertium 0 0 0 0 0 0 0 0 0 0 0 0 R_gnavus 0 0 0 0 0 0 0 0 0 0 0 0 Microbial comp 2 Cl_disporicum 29 11810 10948 14672 0 11349 13978 3942 0 11995 7005 6268 Cl_orbiscindens 0 510 408 764 0 332 545 544 0 310 319 432 Cl_symbiosum 0 559 508 375 0 665 494 450 0 396 639 650 Co_aerofaciens 0 0 0 0 0 0 1172 0 0 0 247 0 E_rectale 0 0 0 0 0 0 0 12 0 0 0 261 L_bacterium_5_1_57FAA 0 972 801 596 0 860 962 844 0 636 1901 1269 B_producta 0 0 0 0 0 0 0 0 0 0 0 0 Cl_innocuum 0 0 0 0 0 0 0 0 0 0 0 0 Cl_mayombei 0 0 0 0 0 0 0 0 0 0 0 0

TABLE TAC Population of Clades on Days 2, 3 and 4 following dosing with Microbial Compositions 1 × 10⁹ per OTU 1 × 10⁸ per OTU 1 × 10⁷ per OTU −1 2 3 4 −1 2 3 4 −1 2 3 4 Microbial comp 1 clade_252 0 444 252 87 0 198 122 125 209 394 231 88 clade_253 10 1746 1190 887 0 1746 769 1011 201 11175 1531 1152 clade_260 0 599 594 438 0 554 346 433 0 521 454 390 clade_262 0 14 151 51 0 0 0 0 0 12 21 57 clade_309 0 11093 9750 4023 0 9991 5208 5145 19 9311 6369 4951 clade_351 0 9064 10647 7751 0 6528 7259 8213 0 8903 10049 8701 clade_354 0 0 0 0 0 0 0 31 173 0 0 0 clade_360 0 14300 10220 11036 0 12553 12989 6889 0 9308 13483 9292 clade_408 13 8892 12985 12101 23 3952 7260 10652 43 4079 8581 14929 clade_494 0 226 227 565 0 188 184 411 0 221 200 351 clade_537 0 0 68 225 0 0 0 0 0 0 0 55 clade_553 0 0 0 0 0 0 0 0 0 0 0 0 Microbial comp 2 clade_253 29 11810 10948 14672 0 11349 13978 3942 0 11995 7005 6268 clade_260 0 1125 1312 854 0 1049 1295 1250 0 792 2121 1637 clade_309 54 12513 13731 7849 0 11610 12004 12672 0 7407 14111 10858 clade_351 0 7651 9939 5936 0 8495 9724 9207 0 6005 9833 7655 clade_354 149 0 127 429 0 0 0 39 12 0 0 0 clade_408 18 2242 4989 10480 12 1688 5580 3789 0 1068 1561 6281 clade_444 41 0 49 202 0 18 0 12 0 14 82 1578 clade_494 0 510 465 1054 0 332 565 596 0 310 319 476 clade_553 0 0 0 0 0 0 1172 0 0 0 247 0

Upon examining the OTU data in Table TAB, several patterns emerge. First, there are a group of OTUs with no sequence reads on Day −1 that show subsequent and large numbers of sequence reads on Days 2, 3, or 4; this group includes Cl. butyricum, Cl. hylemonae, Cl. orbiscindens, Cl. symbiosum, and L. bacterium_5_1_57FAA. Cl. disporicum is comparable to this group as it has sequence reads on Day −1 that are very close to background (10 and 29 in compositions 1 and 2, respectively), which subsequently increase by as much as 1000-fold on Days 2, 3 or 4. Second, there are OTUs such as Co. aerofaciens, C. comes, R. bromii, B. producta, Cl. bolteae, Cl. mayombei, Cl. innocuum, Cl. tertium and R. gnavus which are not detectable at the OTU level in either the Day −1 sample or in subsequent samples. In composition 2, Co. aerofaciens is detected transiently on Day 2 in the 1×10⁸ and 1×10⁷ dose groups; E. rectale in the same experimental groups is detected on Day 3, suggesting a possible relationship between transient population by Co. aerofaciens followed by E. rectale in these groups of mice. A striking observation is that the observed number of OTU sequence reads is not highly dose dependent. Overall, the data is consistent with a model whereby OTUs populate rapidly following oral administration.

The clade-based analysis in Table TAC was performed to more thoroughly evaluate the population of the GI tract. Clade-based analysis obscures some of the details afforded by an OTU analysis. For instance, Cl. tertium and Cl. butyricum are members of the same clade and thus a clade-based analysis cannot distinguish the dynamics of these individual OTUs. However, clade-based analysis has the compensatory benefit that it is sensitive to measuring population changes that can be missed by an OTU-based analysis. The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolving power of the 16S-V4 region for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation. So in some cases, the population of a species can be followed using clade-based assignments when OTU based-detection is insensitive in a complex population. For instance, the clade-based analysis in Table 2B supports the case that R. bromii, B. producta, Cl. innocuum, and R. gnavus were able to populate since each OTU is a sole member of a clade in the microbial compositions and sequence reads went from undetectable on Day −1 to well above background on Days 2, 3 or 4. 16S V4 sequencing and clade-based analysis could not determine whether Cl. tertium or Cl. bolteae populated due to the fact that other members of their clades (Cl. butyricum and Cl. symbiosum, respectively) were present and shown to populate at the OTU level in the mice.

In the mice challenged in parallel with C. difficile, animals were significantly protected as shown in Table TAD. Mice gavaged with vehicle (phosphate buffered saline) experienced 100% mortality while microbial compositions 1 and 2 protected at all dose levels with between 0 and 10% mortality by Day 6, the last day of the experiment. In addition, weight loss in animals treated with microbial compositions 1 and 2 was minimal compared to animals receiving the vehicle gavage. These data confirm that population of the gastrointestinal tract with microbial compositions confers a clinical benefit by restoring a state of dysbiosis so that animals can resist infection by a pathogen.

TABLE TAD Mortality by experimental group in mice challenged with 10^(4.5) C. difficile spores on Day 0 Group Dose (CFU per OTU) Deaths (% mortality) Vehicle control N/A 10 (100%) Microbial composition 1 109 1 (10%) 108 1 (10%) 107 0 (0%) Microbial composition 2 109 0 (0%) 108 1 (10%) 107 1 (10%)

Example 36: Prophylactic Use and Treatment in a Mouse Model of Vancomycin Resistant Enterococcus (VRE) Colonization

The emergence and spread of highly antibiotic-resistant bacteria represent a major clinical challenge (Snitkin et al Science Translational Medicine, 2012). In recent years, the numbers of infections caused by organisms such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus (VRE), and Clostridium difficile have increased markedly, and many of these strains are acquiring resistance to the few remaining active antibiotics. Most infections produced by highly antibiotic-resistant bacteria are acquired during hospitalizations, and preventing patient-to-patient transmission of these pathogens is one of the major challenges confronting hospitals and clinics. Most highly antibiotic-resistant bacterial strains belong to genera that colonize mucosal surfaces, usually at low densities. The highly complex microbiota that normally colonizes mucosal surfaces inhibits expansion of and domination by bacteria such as Enterobacteriaceae and Enterococcaceae. Destruction of the normal flora by antibiotic administration, however, disinhibition antibiotic-resistant members of these bacterial families, leading to their expansion to very high densities (Ubeda et al Journal of Clinical Investigation 2010). High-density colonization by these organisms can be calamitous for the susceptible patient, resulting in bacteremia and sepsis (Taur et al, Clinical Infectious Disease, 2012).

To test prophylactic use and treatment of a bacterial composition test article e.g. spore population, a VRE infection mouse model is used as previously described (Ubeda et al, Infectious Immunity 2013, Ubeda et al, Journal of clinical investigation, 2010). Briefly, experiments are done with 7-week-old C57BL/6J female mice purchased from Jackson Laboratory, housed with irradiated food, and provided with acidified water. Mice are individually housed to avoid contamination between mice due to coprophagia. For experimental infections with VRE, mice are treated with ampicillin (0.5 g/liter) in their drinking water, which is changed every 3 days.

In the treatment model, on day 1, mice are infected by means of oral gavage with 10⁸ CFU of the vancomycin-resistant Enterococcus faecium strain purchased from ATCC (ATCC 700221). One day after infection (day 1), antibiotic treatment is stopped and VRE levels are determined at different time points by plating serial dilutions of fecal pellets on Enterococcosel agar plates (Difco) with vancomycin (8 ug/ml; Sigma). VRE colonies are identified by appearance and confirmed by Gram staining or other methods previously described (e.g. see example 1, 2 and 3). In addition, as previously described (Ubeda et al Journal of Clinical Investigation 2010), PCR of the vanA gene, which confers resistance to vancomycin, confirms the presence of VRE in infected mice. The test article e.g. bacterial composition, or ethanol treated, gradient purified spore preparation (as described herein), fecal suspension, or antibiotic treatment is delivered in PBS on days 1-3 while the negative control contains only PBS and is also delivered on days 1-3 by oral gavage. Fresh fecal stool pellets are obtained daily for the duration of the experiment from days −7 to day 10. The samples are immediately frozen and stored at −80° C. DNA was extracted using standard techniques and analyzed with 16S or comparable methods (e.g. see example 2 and 3).

In the colonization model, ampicillin is administered as described above for day −7 to day 1, treatment with the test article or vehicle control is administered on day 0-2 and the VRE resistant bacteria at 10⁸ CFU are administered on day 14. Fecal samples are taken throughout the experiment daily from −7 to day 21 and submitted for 16S sequencing as previously described (e.g. see examples 2 and 3).

In both models titers of VRE in feces are used to evaluate the success of the test article versus the negative control. Furthermore, microbiota composition is assessed for the ability of the test article to induce a healthy microbiome.

Example 37: Prophylactic Use and Treatment of a Mouse Model of Carbapenem Resistant Klebsiella (CRKB) Colonization

The emergence of Klebsiella pneumoniae strains with decreased susceptibility to carbapenems is a significant threat to hospitalized patients. Resistance to carbapenems in these organisms is most frequently mediated by K. pneumoniae carbapenemase (KPC), a class A beta-lactamase that also confers resistance to broad-spectrum cephalosporins and commercially available beta-lactam/beta-lactamase inhibitor combinations (Queenan et al, Clinical Microbiology Review, 2007). KPC-producing K. pneumoniae (KPC-Kp) strains often harbor resistance determinants against several other classes of antimicrobials, including aminoglycosides and fluoroquinolones, resulting in truly multidrug-resistant (MDR) organisms (Hirsch et al, Journal of Antimicrobial Chemotherapy, 2009). Considering the limited antimicrobial options, infections caused by KPC-Kp pose a tremendous therapeutic challenge and are associated with poor clinical outcomes

A treatment protocol in a mouse model as previously described (e.g. Perez et al, Antimicrobial Agents Chemotherapy, 2011) is used to evaluate the test article e.g. bacterial composition for treating carbapenem resistant Klebsiella and reducing carriage in the GI tract. Female CF1 mice (Harlan Sprague-Dawley, Indianapolis, Ind.) are used and are individually housed and weighed between 25 and 30 g.

The thoroughly characterized strain of K. pneumoniae, VA-367 (8, 9, 25) is used in this study. This clinical isolate is genetically related to the KPC-Kp strain circulating in the Eastern United States. Characterization of the resistance mechanisms in K. pneumoniae VA-367 with PCR and DNA sequence analysis revealed the presence of blaKpc-3, blaTEM-1, blasHv-11, and blasHv-12 as well as qnrB19 and aac(6′)-lb. Additionally, PCR and DNA sequencing revealed disruptions in the coding sequences of the following outer membrane protein genes: ompK35, ompK36, and ompK37. Antibiotic susceptibility testing (AST) was performed with the agar dilution method and interpreted according to current recommendations from the Clinical and Laboratory Standards Institute (CLSI). A modified Hodge test was performed, according to a method described previously (e.g. see Anderson et al, Journal of Clinical Microbiology, 2007) with ertapenem, meropenem, and imipenem. Tigecycline and polymyxin E were evaluated by Etest susceptibility assays (AB bioM'erieux, Solna, Sweden). Results for tigecycline were interpreted as suggested by the U.S. Food and Drug Administration (FDA) and according to CLSI recommendations (criteria for Pseudomonas) for polymyxin E.

Mice (10 per group) are assigned to either a test article e.g. bacterial composition, ethanol treated, spore preparation (e.g. see example 6), antibiotic clindamycin, piperacillin-tazobactam, tigecycline, ertapenem, cefepime, ciprofloxacin, or combination thereof or control group receiving only the vehicle. They are administered the test article daily from day −10 to day 0, On day 0, 10³ CFU of KPC-Kp VA-367 diluted in 0.5 ml phosphate-buffered saline (PBS) was administered by oral gavage using a stainless-steel feeding tube (Perfektum; Popper & Sons, New Hyde Park, N.Y.). Stool samples were collected 1, 4, 6, and 11 days after the administration of KPC-Kp in order to measure the concentration of carbapenem-resistant K. pneumoniae. Stool samples (100 mg diluted in 800 ml of PBS) are plated onto MacConkey agar with and without 0.5 ug/ml of imipenem, and the number of CFU per gram of stool was determined. Alternatively other methods may be used to measure the levels of carbapenem-resistant K. pneumoniae e.g. per, antigen testing, as one who's skilled in the art could perform.

Stool samples were collected after 5 days of treatment to assess the effects of the antibiotics on the stool microflora and to measure antibiotic levels in stool. To assess the effects on the microflora, fresh stool samples as previously described (e.g. see examples 2 and 3). Additional experiments are performed to examine whether the administration the test article e.g. bacterial composition resulted in the elimination or persistence of colonization with KPC-Kp VA-367.

Mice are treated with subcutaneous clindamycin to reduce the normal intestinal flora 1 day before receiving 10⁴ CFU of KPC-Kp VA-367 by oral gavage, and the mice continued to receive subcutaneous clindamycin every other day for 7 days. Concurrently, for 7 days after oral gavage with KPC-Kp, mice received oral gavage of normal saline (control group), or the bacterial composition as specified. An additional dose of subcutaneous clindamycin was administered 20 days after the administration of KPC-Kp VA-367 to assess whether low levels of carbapenem-resistant K. pneumoniae were present that could be augmented by the elimination of the anaerobic microflora. Stool samples were collected at baseline and at 3, 6, 8, 11, 16, and 21 days after KPC-Kp VA-367 was given by gavage. The bacterial composition will be examined by the reduction of CRKB in feces.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series.

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

ADDITIONAL TABLES

Clade membership of bacterial OTUs is based on 16S sequence data. Clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood methods familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another, and (ii) within 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data, while OTUs falling within the same clade are closely related. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. Members of the same clade, due to their evolutionary relatedness, play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention. All OTUs are denoted as to their putative capacity to form spores and whether they are a Pathogen or Pathobiont (see Definitions for description of “Pathobiont”). NIAID Priority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or ‘Category-C’, and Opportunistic Pathogens are denoted as “OP”. OTUs that are not pathogenic or for which their ability to exist as a pathogen is unknown are denoted as “N”. The ‘SEQ ID Number’ denotes the identifier of the OTU in the Sequence Listing File and ‘Public DB Accession’ denotes the identifier of the OTU in a public sequence repository.

TABLE 1 List of Operational Taxonomic Units (OTU) with taxonomic assignments made to Genus, Species, and Phylogenetic Clade SEQ ID Public DB Spore Pathogen OTU Number Accession Clade Former Status Eubacterium saburreum 858 AB525414 clade_178 Y N Eubacterium sp. oral clone 866 AY349376 clade_178 Y N IR009 Lachnospiraceae bacterium 1061 HQ616401 clade_178 Y N ICM62 Lachnospiraceae bacterium 1062 HQ616384 clade_178 Y N MSX33 Lachnospiraceae bacterium 1063 ADDS01000069 clade_178 Y N oral taxon 107 Alicyclobacillus acidocaldarius 122 NR_074721 clade_179 Y N Clostridium baratii 555 NR_029229 clade_223 Y N Clostridium colicanis 576 FJ957863 clade_223 Y N Clostridium paraputrificum 611 AB536771 clade_223 Y N Clostridium sardiniense 621 NR_041006 clade_223 Y N Eubacterium budayi 837 NR_024682 clade_223 Y N Eubacterium moniliforme 851 HF558373 clade_223 Y N Eubacterium multiforme 852 NR_024683 clade_223 Y N Eubacterium nitritogenes 853 NR_024684 clade_223 Y N Anoxybacillus flavithermus 173 NR_074667 clade_238 Y N Bacillus aerophilus 196 NR_042339 clade_238 Y N Bacillus aestuarii 197 GQ980243 clade_238 Y N Bacillus amyloliquefaciens 199 NR_075005 clade_238 Y N Bacillus anthracis 200 AAEN01000020 clade_238 Y Category-A Bacillus atrophaeus 201 NR_075016 clade_238 Y OP Bacillus badius 202 NR_036893 clade_238 Y OP Bacillus cereus 203 ABDJ01000015 clade_238 Y OP Bacillus circulans 204 AB271747 clade_238 Y OP Bacillus firmus 207 NR_025842 clade_238 Y OP Bacillus flexus 208 NR_024691 clade_238 Y OP Bacillus fordii 209 NR_025786 clade_238 Y OP Bacillus halmapalus 211 NR_026144 clade_238 Y OP Bacillus herbersteinensis 213 NR_042286 clade_238 Y OP Bacillus idriensis 215 NR_043268 clade_238 Y OP Bacillus lentus 216 NR_040792 clade_238 Y OP Bacillus licheniformis 217 NC_006270 clade_238 Y OP Bacillus megaterium 218 GU252124 clade_238 Y OP Bacillus nealsonii 219 NR_044546 clade_238 Y OP Bacillus niabensis 220 NR_043334 clade_238 Y OP Bacillus niacini 221 NR_024695 clade_238 Y OP Bacillus pocheonensis 222 NR_041377 clade_238 Y OP Bacillus pumilus 223 NR_074977 clade_238 Y OP Bacillus safensis 224 JQ624766 clade_238 Y OP Bacillus simplex 225 NR_042136 clade_238 Y OP Bacillus sonorensis 226 NR_025130 clade_238 Y OP Bacillus sp. 10403023 227 CAET01000089 clade_238 Y OP MM10403188 Bacillus sp. 2_A_57_CT2 230 ACWD01000095 clade_238 Y OP Bacillus sp. 2008724126 228 GU252108 clade_238 Y OP Bacillus sp. 2008724139 229 GU252111 clade_238 Y OP Bacillus sp. 7_16AIA 231 FN397518 clade_238 Y OP Bacillus sp. AP8 233 JX101689 clade_238 Y OP Bacillus sp. B27(2008) 234 EU362173 clade_238 Y OP Bacillus sp. BT1B_CT2 235 ACWC01000034 clade_238 Y OP Bacillus sp. GB1.1 236 FJ897765 clade_238 Y OP Bacillus sp. GB9 237 FJ897766 clade_238 Y OP Bacillus sp. HU19.1 238 FJ897769 clade_238 Y OP Bacillus sp. HU29 239 FJ897771 clade_238 Y OP Bacillus sp. HU33.1 240 FJ897772 clade_238 Y OP Bacillus sp. JC6 241 JF824800 clade_238 Y OP Bacillus sp. oral taxon F79 248 HM099654 clade_238 Y OP Bacillus sp. SRC_DSF1 243 GU797283 clade_238 Y OP Bacillus sp. SRC_DSF10 242 GU797292 clade_238 Y OP Bacillus sp. SRC_DSF2 244 GU797284 clade_238 Y OP Bacillus sp. SRC_DSF6 245 GU797288 clade_238 Y OP Bacillus sp. tc09 249 HQ844242 clade_238 Y OP Bacillus sp. zh168 250 FJ851424 clade_238 Y OP Bacillus sphaericus 251 DQ286318 clade_238 Y OP Bacillus sporothermodurans 252 NR_026010 clade_238 Y OP Bacillus subtilis 253 EU627588 clade_238 Y OP Bacillus thermoamylovorans 254 NR_029151 clade_238 Y OP Bacillus thuringiensis 255 NC_008600 clade_238 Y OP Bacillus weihenstephanensis 256 NR_074926 clade_238 Y OP Geobacillus kaustophilus 933 NR_074989 clade_238 Y N Geobacillus stearothermophilus 936 NR_040794 clade_238 Y N Geobacillus thermodenitrificans 938 NR_074976 clade_238 Y N Geobacillus thermoglucosidasius 939 NR_043022 clade_238 Y N Lysinibacillus sphaericus 1193 NR_074883 clade_238 Y N Clostridiales sp. SS3_4 543 AY305316 clade_246 Y N Clostridium beijerinckii 557 NR_074434 clade_252 Y N Clostridium botulinum 560 NC_010723 clade_252 Y Category-A Clostridium butyricum 561 ABDT01000017 clade_252 Y N Clostridium chauvoei 568 EU106372 clade_252 Y N Clostridium favososporum 582 X76749 clade_252 Y N Clostridium histolyticum 592 HF558362 clade_252 Y N Clostridium isatidis 597 NR_026347 clade_252 Y N Clostridium limosum 602 FR870444 clade_252 Y N Clostridium sartagoforme 622 NR_026490 clade_252 Y N Clostridium septicum 624 NR_026020 clade_252 Y N Clostridium sp. 7_2_43FAA 626 ACDK01000101 clade_252 Y N Clostridium sporogenes 645 ABKW02000003 clade_252 Y N Clostridium tertium 653 Y18174 clade_252 Y N Clostridium carnis 564 NR_044716 clade_253 Y N Clostridium celatum 565 X77844 clade_253 Y N Clostridium disporicum 579 NR_026491 clade_253 Y N Clostridium gasigenes 585 NR_024945 clade_253 Y N Clostridium quinii 616 NR_026149 clade_253 Y N Clostridium hylemonae 593 AB023973 clade_260 Y N Clostridium scindens 623 AF262238 clade_260 Y N Lachnospiraceae bacterium 1054 ACTR01000020 clade_260 Y N 5_1_57FAA Clostridium glycyrrhizinilyticum 588 AB233029 clade_262 Y N Clostridium nexile 607 X73443 clade_262 Y N Coprococcus comes 674 ABVR01000038 clade_262 Y N Lachnospiraceae bacterium 1048 ACTM01000065 clade_262 Y N 1_1_57FAA Lachnospiraceae bacterium 1049 ACTN01000028 clade_262 Y N 1_4_56FAA Lachnospiraceae bacterium 1057 ACWQ01000079 clade_262 Y N 8_1_57FAA Ruminococcus lactaris 1663 ABOU02000049 clade_262 Y N Ruminococcus torques 1670 AAVP02000002 clade_262 Y N Paenibacillus lautus 1397 NR_040882 clade_270 Y N Paenibacillus polymyxa 1399 NR_037006 clade_270 Y N Paenibacillus sp. HGF5 1402 AEXS01000095 clade_270 Y N Paenibacillus sp. HGF7 1403 AFDH01000147 clade_270 Y N Eubacterium sp. oral clone 868 AY349379 clade_298 Y N JI012 Alicyclobacillus contaminans 124 NR_041475 clade_301 Y N Alicyclobacillus herbarius 126 NR_024753 clade_301 Y N Alicyclobacillus pomorum 127 NR_024801 clade_301 Y N Blautia coccoides 373 AB571656 clade_309 Y N Blautia glucerasea 374 AB588023 clade_309 Y N Blautia glucerasei 375 AB439724 clade_309 Y N Blautia hansenii 376 ABYU02000037 clade_309 Y N Blautia luti 378 AB691576 clade_309 Y N Blautia producta 379 AB600998 clade_309 Y N Blautia schinkii 380 NR_026312 clade_309 Y N Blautia sp. M25 381 HM626178 clade_309 Y N Blautia stercoris 382 HM626177 clade_309 Y N Blautia wexlerae 383 EF036467 clade_309 Y N Bryantella formatexigens 439 ACCL02000018 clade_309 Y N Clostridium coccoides 573 EF025906 clade_309 Y N Eubacterium cellulosolvens 839 AY178842 clade_309 Y N Lachnospiraceae bacterium 1056 ACTV01000014 clade_309 Y N 6_1_63FAA Ruminococcus hansenii 1662 M59114 clade_309 Y N Ruminococcus obeum 1664 AY169419 clade_309 Y N Ruminococcus sp. 1666 ACII01000172 clade_309 Y N 5_1_39BFAA Ruminococcus sp. K_1 1669 AB222208 clade_309 Y N Syntrophococcus sucromutans 1911 NR_036869 clade_309 Y N Bacillus alcalophilus 198 X76436 clade_327 Y N Bacillus clausii 205 FN397477 clade_327 Y OP Bacillus gelatini 210 NR_025595 clade_327 Y OP Bacillus halodurans 212 AY144582 clade_327 Y OP Bacillus sp. oral taxon F26 246 HM099642 clade_327 Y OP Clostridium innocuum 595 M23732 clade_351 Y N Clostridium sp. HGF2 628 AENW01000022 clade_351 Y N Clostridium perfringens 612 ABDW01000023 clade_353 Y Category-B Sarcina ventriculi 1687 NR_026146 clade_353 Y N Clostridium bartlettii 556 ABEZ02000012 clade_354 Y N Clostridium bifermentans 558 X73437 clade_354 Y N Clostridium ghonii 586 AB542933 clade_354 Y N Clostridium glycolicum 587 FJ384385 clade_354 Y N Clostridium mayombei 605 FR733682 clade_354 Y N Clostridium sordellii 625 AB448946 clade_354 Y N Clostridium sp. MT4 E 635 FJ159523 clade_354 Y N Eubacterium tenue 872 M59118 clade_354 Y N Clostridium argentinense 553 NR_029232 clade_355 Y N Clostridium sp. JC122 630 CAEV01000127 clade_355 Y N Clostridium sp. NMBHI_1 636 JN093130 clade_355 Y N Clostridium subterminale 650 NR_041795 clade_355 Y N Clostridium sulfidigenes 651 NR_044161 clade_355 Y N Dorea formicigenerans 773 AAXA02000006 clade_360 Y N Dorea longicatena 774 AJ132842 clade_360 Y N Lachnospiraceae bacterium 1050 ADLB01000035 clade_360 Y N 2_1_46FAA Lachnospiraceae bacterium 1051 ACTO01000052 clade_360 Y N 2_1_58FAA Lachnospiraceae bacterium 1053 ADCR01000030 clade_360 Y N 4_1_37FAA Lachnospiraceae bacterium 1058 ACTX01000023 clade_360 Y N 9_1_43BFAA Ruminococcus gnavus 1661 X94967 clade_360 Y N Ruminococcus sp. ID8 1668 AY960564 clade_360 Y N Blautia hydrogenotrophica 377 ACBZ01000217 clade_368 Y N Lactonifactor longoviformis 1147 DQ100449 clade_368 Y N Robinsoniella peoriensis 1633 AF445258 clade_368 Y N Eubacterium infirmum 849 U13039 clade_384 Y N Eubacterium sp. WAL 14571 864 FJ687606 clade_384 Y N Erysipelotrichaceae bacterium 823 ACZW01000054 clade_385 Y N 5_2_54FAA Eubacterium biforme 835 ABYT01000002 clade_385 Y N Eubacterium cylindroides 842 FP929041 clade_385 Y N Eubacterium dolichum 844 L34682 clade_385 Y N Eubacterium sp. 3_1_31 861 ACTL01000045 clade_385 Y N Eubacterium tortuosum 873 NR_044648 clade_385 Y N Bulleidia extructa 441 ADFR01000011 clade_388 Y N Solobacterium moorei 1739 AECQ01000039 clade_388 Y N Coprococcus catus 673 EU266552 clade_393 Y N Lachnospiraceae bacterium 1064 HM099641 clade_393 Y N oral taxon F15 Clostridium cochlearium 574 NR_044717 clade_395 Y N Clostridium malenominatum 604 FR749893 clade_395 Y N Clostridium tetani 654 NC_004557 clade_395 Y N Acetivibrio ethanolgignens 6 FR749897 clade_396 Y N Anaerosporobacter mobilis 161 NR_042953 clade_396 Y N Bacteroides pectinophilus 288 ABVQ01000036 clade_396 Y N Clostridium aminovalericum 551 NR_029245 clade_396 Y N Clostridium phytofermentans 613 NR_074652 clade_396 Y N Eubacterium hallii 848 L34621 clade_396 Y N Eubacterium xylanophilum 875 L34628 clade_396 Y N Ruminococcus callidus 1658 NR_029160 clade_406 Y N Ruminococcus champanellensis 1659 FP929052 clade_406 Y N Ruminococcus sp. 18P13 1665 AJ515913 clade_406 Y N Ruminococcus sp. 9SE51 1667 FM954974 clade_406 Y N Anaerostipes caccae 162 ABAX03000023 clade_408 Y N Anaerostipes sp. 3_2_56FAA 163 ACWB01000002 clade_408 Y N Clostridiales bacterium 541 ABQR01000074 clade_408 Y N 1_7_47FAA Clostridiales sp. SM4_1 542 FP929060 clade_408 Y N Clostridiales sp. SSC_2 544 FP929061 clade_408 Y N Clostridium aerotolerans 546 X76163 clade_408 Y N Clostridium aldenense 547 NR_043680 clade_408 Y N Clostridium algidixylanolyticum 550 NR_028726 clade_408 Y N Clostridium amygdalinum 552 AY353957 clade_408 Y N Clostridium asparagiforme 554 ACCJ01000522 clade_408 Y N Clostridium bolteae 559 ABCC02000039 clade_408 Y N Clostridium celerecrescens 566 JQ246092 clade_408 Y N Clostridium citroniae 569 ADLJ01000059 clade_408 Y N Clostridium clostridiiformes 571 M59089 clade_408 Y N Clostridium clostridioforme 572 NR_044715 clade_408 Y N Clostridium hathewayi 590 AY552788 clade_408 Y N Clostridium indolis 594 AF028351 clade_408 Y N Clostridium lavalense 600 EF564277 clade_408 Y N Clostridium saccharolyticum 620 CP002109 clade_408 Y N Clostridium sp. M62_1 633 ACFX02000046 clade_408 Y N Clostridium sp. SS2_1 638 ABGC03000041 clade_408 Y N Clostridium sphenoides 643 X73449 clade_408 Y N Clostridium symbiosum 652 ADLQ01000114 clade_408 Y N Clostridium xylanolyticum 658 NR_037068 clade_408 Y N Eubacterium hadrum 847 FR749933 clade_408 Y N Lachnospiraceae bacterium 1052 ACTP01000124 clade_408 Y N 3_1_57FAA_CT1 Lachnospiraceae bacterium 1055 ACTS01000081 clade_408 Y N 5_1_63FAA Lachnospiraceae bacterium A4 1059 DQ789118 clade_408 Y N Lachnospiraceae bacterium 1060 EU728771 clade_408 Y N DJF VP30 Lachnospiraceae genomosp. 1065 AY278618 clade_408 Y N C1 Clostridium difficile 578 NC_013315 clade_409 Y OP Eubacterium sp. AS15b 862 HQ616364 clade_428 Y N Eubacterium sp. OBRC9 863 HQ616354 clade_428 Y N Eubacterium sp. oral clone 871 AY947497 clade_428 Y N OH3A Eubacterium yurii 876 AEES01000073 clade_428 Y N Clostridium acetobutylicum 545 NR_074511 clade_430 Y N Clostridium algidicarnis 549 NR_041746 clade_430 Y N Clostridium cadaveris 562 AB542932 clade_430 Y N Clostridium carboxidivorans 563 FR733710 clade_430 Y N Clostridium estertheticum 580 NR_042153 clade_430 Y N Clostridium fallax 581 NR_044714 clade_430 Y N Clostridium felsineum 583 AF270502 clade_430 Y N Clostridium frigidicarnis 584 NR_024919 clade_430 Y N Clostridium kluyveri 598 NR_074165 clade_430 Y N Clostridium magnum 603 X77835 clade_430 Y N Clostridium putrefaciens 615 NR_024995 clade_430 Y N Clostridium sp. HPB_46 629 AY862516 clade_430 Y N Clostridium tyrobutyricum 656 NR_044718 clade_430 Y N Sutterella parvirubra 1899 AB300989 clade_432 Y N Acetanaerobacterium 4 NR_042930 clade_439 Y N elongatum Clostridium cellulosi 567 NR_044624 clade_439 Y N Ethanoligenens harbinense 832 AY675965 clade_439 Y N Eubacterium rectale 856 FP929042 clade_444 Y N Eubacterium sp. oral clone 865 AY349374 clade_444 Y N GI038 Lachnobacterium bovis 1045 GU324407 clade_444 Y N Roseburia cecicola 1634 GU233441 clade_444 Y N Roseburia faecalis 1635 AY804149 clade_444 Y N Roseburia faecis 1636 AY305310 clade_444 Y N Roseburia hominis 1637 AJ270482 clade_444 Y N Roseburia intestinalis 1638 FP929050 clade_444 Y N Roseburia inulinivorans 1639 AJ270473 clade_444 Y N Brevibacillus brevis 410 NR_041524 clade_448 Y N Brevibacillus laterosporus 414 NR_037005 clade_448 Y N Bacillus coagulans 206 DQ297928 clade_451 Y OP Sporolactobacillus inulinus 1752 NR_040962 clade_451 Y N Kocuria palustris 1041 EU333884 clade_453 Y N Nocardia farcinica 1353 NC_006361 clade_455 Y N Bacillus sp. oral taxon F28 247 HM099650 clade_456 Y OP Catenibacterium mitsuokai 495 AB030224 clade_469 Y N Clostridium sp. TM_40 640 AB249652 clade_469 Y N Coprobacillus cateniformis 670 AB030218 clade_469 Y N Coprobacillus sp. 29_1 671 ADKX01000057 clade_469 Y N Clostridium rectum 618 NR_029271 clade_470 Y N Eubacterium nodatum 854 U13041 clade_476 Y N Eubacterium saphenum 859 NR_026031 clade_476 Y N Eubacterium sp. oral clone 867 AY349373 clade_476 Y N JH012 Eubacterium sp. oral clone 870 AY349378 clade_476 Y N JS001 Faecalibacterium prausnitzii 880 ACOP02000011 clade_478 Y N Gemmiger formicilis 932 GU562446 clade_478 Y N Subdoligranulum variabile 1896 AJ518869 clade_478 Y N Clostridiaceae bacterium JC13 532 JF824807 clade_479 Y N Clostridium sp. MLG055 634 AF304435 clade_479 Y N Erysipelotrichaceae bacterium 822 ACTJ01000113 clade_479 Y N 3_1_53 Clostridium cocleatum 575 NR_026495 clade_481 Y N Clostridium ramosum 617 M23731 clade_481 Y N Clostridium saccharogumia 619 DQ100445 clade_481 Y N Clostridium spiroforme 644 X73441 clade_481 Y N Coprobacillus sp. D7 672 ACDT01000199 clade_481 Y N Clostridiales bacterium 535 AB477431 clade_482 Y N SY8519 Clostridium sp. SY8519 639 AP012212 clade_482 Y N Eubacterium ramulus 855 AJ011522 clade_482 Y N Erysipelothrix inopinata 819 NR_025594 clade_485 Y N Erysipelothrix rhusiopathiae 820 ACLK01000021 clade_485 Y N Erysipelothrix tonsillarum 821 NR_040871 clade_485 Y N Holdemania filiformis 1004 Y11466 clade_485 Y N Mollicutes bacterium pACH93 1258 AY297808 clade_485 Y N Coxiella burnetii 736 CP000890 clade_486 Y Category-B Clostridium hiranonis 591 AB023970 clade_487 Y N Clostridium irregulare 596 NR_029249 clade_487 Y N Clostridium orbiscindens 609 Y18187 clade_494 Y N Clostridium sp. NML 04A032 637 EU815224 clade_494 Y N Flavonifractor plautii 886 AY724678 clade_494 Y N Pseudoflavonifractor capillosus 1591 AY136666 clade_494 Y N Ruminococcaceae bacterium 1655 ADDX01000083 clade_494 Y N D16 Acetivibrio cellulolyticus 5 NR_025917 clade_495 Y N Clostridium aldrichii 548 NR_026099 clade_495 Y N Clostridium clariflavum 570 NR_041235 clade_495 Y N Clostridium stercorarium 647 NR_025100 clade_495 Y N Clostridium straminisolvens 649 NR_024829 clade_495 Y N Clostridium thermocellum 655 NR_074629 clade_495 Y N Fusobacterium nucleatum 901 ADVK01000034 clade_497 Y N Eubacterium barkeri 834 NR_044661 clade_512 Y N Eubacterium callanderi 838 NR_026330 clade_512 Y N Eubacterium limosum 850 CP002273 clade_512 Y N Anaerotruncus colihominis 164 ABGD02000021 clade_516 Y N Clostridium methylpentosum 606 ACEC01000059 clade_516 Y N Clostridium sp. YIT 12070 642 AB491208 clade_516 Y N Hydrogenoanaerobacterium 1005 NR_044425 clade_516 Y N saccharovorans Ruminococcus albus 1656 AY445600 clade_516 Y N Ruminococcus flavefaciens 1660 NR_025931 clade_516 Y N Clostridium haemolyticum 589 NR_024749 clade_517 Y N Clostridium novyi 608 NR_074343 clade_517 Y N Clostridium sp. LMG 16094 632 X95274 clade_517 Y N Eubacterium ventriosum 874 L34421 clade_519 Y N Bacteroides galacturonicus 280 DQ497994 clade_522 Y N Eubacterium eligens 845 CP001104 clade_522 Y N Lachnospira multipara 1046 FR733699 clade_522 Y N Lachnospira pectinoschiza 1047 L14675 clade_522 Y N Lactobacillus rogosae 1114 GU269544 clade_522 Y N Bacillus horti 214 NR_036860 clade_527 Y OP Bacillus sp. 9_3AIA 232 FN397519 clade_527 Y OP Eubacterium brachy 836 U13038 clade_533 Y N Filifactor alocis 881 CP002390 clade_533 Y N Filifactor villosus 882 NR_041928 clade_533 Y N Clostridium leptum 601 AJ305238 clade_537 Y N Clostridium sp. YIT 12069 641 AB491207 clade_537 Y N Clostridium sporosphaeroides 646 NR_044835 clade_537 Y N Eubacterium coprostanoligenes 841 HM037995 clade_537 Y N Ruminococcus bromii 1657 EU266549 clade_537 Y N Eubacterium siraeum 860 ABCA03000054 clade_538 Y N Clostridium viride 657 NR_026204 clade_540 Y N Oscillibacter sp. G2 1386 HM626173 clade_540 Y N Oscillibacter valericigenes 1387 NR_074793 clade_540 Y N Oscillospira guilliermondii 1388 AB040495 clade_540 Y N Butyrivibrio crossotus 455 ABWN01000012 clade_543 Y N Clostridium sp. L2_50 631 AAYW02000018 clade_543 Y N Coprococcus eutactus 675 EF031543 clade_543 Y N Coprococcus sp. ART55_1 676 AY350746 clade_543 Y N Eubacterium ruminantium 857 NR_024661 clade_543 Y N Collinsella aerofaciens 659 AAVN02000007 clade_553 Y N Alkaliphilus metalliredigenes 137 AY137848 clade_554 Y N Alkaliphilus oremlandii 138 NR_043674 clade_554 Y N Clostridium sticklandii 648 L04167 clade_554 Y N Turicibacter sanguinis 1965 AF349724 clade_555 Y N Fulvimonas sp. NML 060897 892 EF589680 clade_557 Y N Desulfitobacterium frappieri 753 AJ276701 clade_560 Y N Desulfitobacterium hafniense 754 NR_074996 clade_560 Y N Desulfotomaculum nigrificans 756 NR_044832 clade_560 Y N Lutispora thermophila 1191 NR_041236 clade_564 Y N Brachyspira pilosicoli 405 NR_075069 clade_565 Y N Eggerthella lenta 778 AF292375 clade_566 Y N Streptomyces albus 1888 AJ697941 clade_566 Y N Chlamydiales bacterium NS11 505 JN606074 clade_567 Y N Anaerofustis stercorihominis 159 ABIL02000005 clade_570 Y N Butyricicoccus pullicaecorum 453 HH793440 clade_572 Y N Eubacterium desmolans 843 NR_044644 clade_572 Y N Papillibacter cinnamivorans 1415 NR_025025 clade_572 Y N Sporobacter termitidis 1751 NR_044972 clade_572 Y N Deferribacteres sp. oral clone 744 AY349371 clade_575 Y N JV006 Clostridium colinum 577 NR_026151 clade_576 Y N Clostridium lactatifermentans 599 NR_025651 clade_576 Y N Clostridium piliforme 614 D14639 clade_576 Y N Saccharomonospora viridis 1671 X54286 clade_579 Y N Thermobifida fusca 1921 NC_007333 clade_579 Y N Leptospira licerasiae 1164 EF612284 clade_585 Y OP Moorella thermoacetica 1259 NR_075001 clade_590 Y N Thermoanaerobacter 1920 CP000924 clade_590 Y N pseudethanolicus Flexistipes sinusarabici 888 NR_074881 clade_591 Y N Gloeobacter violaceus 942 NR_074282 clade_596 Y N Eubacterium sp. oral clone 869 AY349377 clade_90 Y N JN088 Clostridium oroticum 610 FR749922 clade_96 Y N Clostridium sp. D5 627 ADBG01000142 clade_96 Y N Eubacterium contortum 840 FR749946 clade_96 Y N Eubacterium fissicatena 846 FR749935 clade_96 Y N Corynebacterium coyleae 692 X96497 clade_100 N N Corynebacterium mucifaciens 711 NR_026396 clade_100 N N Corynebacterium ureicelerivorans 733 AM397636 clade_100 N N Corynebacterium appendicis 684 NR_028951 clade_102 N N Corynebacterium genitalium 698 ACLJ01000031 clade_102 N N Corynebacterium glaucum 699 NR_028971 clade_102 N N Corynebacterium imitans 703 AF537597 clade_102 N N Corynebacterium riegelii 719 EU848548 clade_102 N N Corynebacterium sp. 723 HE575405 clade_102 N N L_2012475 Corynebacterium sp. NML 724 GU238409 clade_102 N N 93_0481 Corynebacterium sundsvallense 728 Y09655 clade_102 N N Corynebacterium tuscaniae 730 AY677186 clade_102 N N Prevotella maculosa 1504 AGEK01000035 clade_104 N N Prevotella oris 1513 ADDV01000091 clade_104 N N Prevotella salivae 1517 AB108826 clade_104 N N Prevotella sp. ICM55 1521 HQ616399 clade_104 N N Prevotella sp. oral clone 1528 AY005057 clade_104 N N AA020 Prevotella sp. oral clone GI032 1538 AY349396 clade_104 N N Prevotella sp. oral taxon G70 1558 GU432179 clade_104 N N Prevotella corporis 1491 L16465 clade_105 N N Bacteroides sp. 4_1_36 312 ACTC01000133 clade_110 N N Bacteroides sp. AR20 315 AF139524 clade_110 N N Bacteroides sp. D20 319 ACPT01000052 clade_110 N N Bacteroides sp. F_4 322 AB470322 clade_110 N N Bacteroides uniformis 329 AB050110 clade_110 N N Prevotella nanceiensis 1510 JN867228 clade_127 N N Prevotella sp. oral taxon 299 1548 ACWZ01000026 clade_127 N N Prevotella bergensis 1485 ACKS01000100 clade_128 N N Prevotella buccalis 1489 JN867261 clade_129 N N Prevotella timonensis 1564 ADEF01000012 clade_129 N N Prevotella oralis 1512 AEPE01000021 clade_130 N N Prevotella sp. SEQ072 1525 JN867238 clade_130 N N Leuconostoc carnosum 1177 NR_040811 clade_135 N N Leuconostoc gasicomitatum 1179 FN822744 clade_135 N N Leuconostoc inhae 1180 NR_025204 clade_135 N N Leuconostoc kimchii 1181 NR_075014 clade_135 N N Edwardsiella tarda 777 CP002154 clade_139 N N Photorhabdus asymbiotica 1466 Z76752 clade_139 N N Psychrobacter arcticus 1607 CP000082 clade_141 N N Psychrobacter cibarius 1608 HQ698586 clade_141 N N Psychrobacter cryohalolentis 1609 CP000323 clade_141 N N Psychrobacter faecalis 1610 HQ698566 clade_141 N N Psychrobacter nivimaris 1611 HQ698587 clade_141 N N Psychrobacter pulmonis 1612 HQ698582 clade_141 N N Pseudomonas aeruginosa 1592 AABQ07000001 clade_154 N N Pseudomonas sp. 2_1_26 1600 ACWU01000257 clade_154 N N Corynebacterium confusum 691 Y15886 clade_158 N N Corynebacterium propinquum 712 NR_037038 clade_158 N N Corynebacterium 713 X84258 clade_158 N N pseudodiphtheriticum Bartonella bacilliformis 338 NC_008783 clade_159 N N Bartonella grahamii 339 CP001562 clade_159 N N Bartonella henselae 340 NC_005956 clade_159 N N Bartonella quintana 341 BX897700 clade_159 N N Bartonella tamiae 342 EF672728 clade_159 N N Bartonella washoensis 343 FJ719017 clade_159 N N Brucella abortus 430 ACBJ01000075 clade_159 N Category-B Brucella canis 431 NR_044652 clade_159 N Category-B Brucella ceti 432 ACJD01000006 clade_159 N Category-B Brucella melitensis 433 AE009462 clade_159 N Category-B Brucella microti 434 NR_042549 clade_159 N Category-B Brucella ovis 435 NC_009504 clade_159 N Category-B Brucella sp. 83_13 436 ACBQ01000040 clade_159 N Category-B Brucella sp. BO1 437 EU053207 clade_159 N Category-B Brucella suis 438 ACBK01000034 clade_159 N Category-B Ochrobactrum anthropi 1360 NC_009667 clade_159 N N Ochrobactrum intermedium 1361 ACQA01000001 clade_159 N N Ochrobactrum 1362 DQ365921 clade_159 N N pseudintermedium Prevotella genomosp. C2 1496 AY278625 clade_164 N N Prevotella multisaccharivorax 1509 AFJE01000016 clade_164 N N Prevotella sp. oral clone 1543 AY550997 clade_164 N N IDR_CEC_0055 Prevotella sp. oral taxon 292 1547 GQ422735 clade_164 N N Prevotella sp. oral taxon 300 1549 GU409549 clade_164 N N Prevotella marshii 1505 AEEI01000070 clade_166 N N Prevotella sp. oral clone IK053 1544 AY349401 clade_166 N N Prevotella sp. oral taxon 781 1554 GQ422744 clade_166 N N Prevotella stercorea 1562 AB244774 clade_166 N N Prevotella brevis 1487 NR_041954 clade_167 N N Prevotella ruminicola 1516 CP002006 clade_167 N N Prevotella sp. sp24 1560 AB003384 clade_167 N N Prevotella sp. sp34 1561 AB003385 clade_167 N N Prevotella albensis 1483 NR_025300 clade_168 N N Prevotella copri 1490 ACBX02000014 clade_168 N N Prevotella oulorum 1514 L16472 clade_168 N N Prevotella sp. BI_42 1518 AJ581354 clade_168 N N Prevotella sp. oral clone 1546 AY207050 clade_168 N N P4PB_83 P2 Prevotella sp. oral taxon G60 1557 GU432133 clade_168 N N Prevotella amnii 1484 AB547670 clade_169 N N Bacteroides caccae 268 EU136686 clade_170 N N Bacteroides finegoldii 277 AB222699 clade_170 N N Bacteroides intestinalis 283 ABJL02000006 clade_171 N N Bacteroides sp. XB44A 326 AM230649 clade_171 N N Bifidobacteriaceae genomosp. 345 AY278612 clade_172 N N C1 Bifidobacterium adolescentis 346 AAXD02000018 clade_172 N N Bifidobacterium angulatum 347 ABYS02000004 clade_172 N N Bifidobacterium animalis 348 CP001606 clade_172 N N Bifidobacterium breve 350 CP002743 clade_172 N N Bifidobacterium catenulatum 351 ABXY01000019 clade_172 N N Bifidobacterium dentium 352 CP001750 clade_172 N OP Bifidobacterium gallicum 353 ABXB03000004 clade_172 N N Bifidobacterium infantis 354 AY151398 clade_172 N N Bifidobacterium 355 AB491757 clade_172 N N kashiwanohense Bifidobacterium longum 356 ABQQ01000041 clade_172 N N Bifidobacterium 357 ABXX02000002 clade_172 N N pseudocatenulatum Bifidobacterium pseudolongum 358 NR_043442 clade_172 N N Bifidobacterium scardovii 359 AJ307005 clade_172 N N Bifidobacterium sp. HM2 360 AB425276 clade_172 N N Bifidobacterium sp. HMLN12 361 JF519685 clade_172 N N Bifidobacterium sp. M45 362 HM626176 clade_172 N N Bifidobacterium sp. MSX5B 363 HQ616382 clade_172 N N Bifidobacterium sp. TM_7 364 AB218972 clade_172 N N Bifidobacterium thermophilum 365 DQ340557 clade_172 N N Leuconostoc citreum 1178 AM157444 clade_175 N N Leuconostoc lactis 1182 NR_040823 clade_175 N N Alicyclobacillus acidoterrestris 123 NR_040844 clade_179 N N Alicyclobacillus cycloheptanicus 125 NR_024754 clade_179 N N Acinetobacter baumannii 27 ACYQ01000014 clade_181 N N Acinetobacter calcoaceticus 28 AM157426 clade_181 N N Acinetobactergenomosp. C1 29 AY278636 clade_181 N N Acinetobacter haemolyticus 30 ADMT01000017 clade_181 N N Acinetobacter johnsonii 31 ACPL01000162 clade_181 N N Acinetobacter junii 32 ACPM01000135 clade_181 N N Acinetobacter lwoffii 33 ACPN01000204 clade_181 N N Acinetobacter parvus 34 AIEB01000124 clade_181 N N Acinetobacter schindleri 36 NR_025412 clade_181 N N Acinetobacter sp. 56A1 37 GQ178049 clade_181 N N Acinetobacter sp. CIP 101934 38 JQ638573 clade_181 N N Acinetobacter sp. CIP 102143 39 JQ638578 clade_181 N N Acinetobacter sp. M16_22 41 HM366447 clade_181 N N Acinetobacter sp. RUH2624 42 ACQF01000094 clade_181 N N Acinetobacter sp. SH024 43 ADCH01000068 clade_181 N N Lactobacillus jensenii 1092 ACQD01000066 clade_182 N N Alcaligenes faecalis 119 AB680368 clade_183 N N Alcaligenes sp. CO14 120 DQ643040 clade_183 N N Alcaligenes sp. S3 121 HQ262549 clade_183 N N Oligella ureolytica 1366 NR_041998 clade_183 N N Oligella urethralis 1367 NR_041753 clade_183 N N Eikenella corrodens 784 ACEA01000028 clade_185 N N Kingella denitrificans 1019 AEWV01000047 clade_185 N N Kingella genomosp. P1 oral 1020 DQ003616 clade_185 N N cone MB2_C20 Kingella kingae 1021 AFHS01000073 clade_185 N N Kingella oralis 1022 ACJW02000005 clade_185 N N Kingella sp. oral clone ID059 1023 AY349381 clade_185 N N Neisseria elongata 1330 ADBF01000003 clade_185 N N Neisseria genomosp. P2 oral 1332 DQ003630 clade_185 N N clone MB5_P15 Neisseria sp. oral clone JC012 1345 AY349388 clade_185 N N Neisseria sp. SMC_A9199 1342 FJ763637 clade_185 N N Simonsiella muelleri 1731 ADCY01000105 clade_185 N N Corynebacterium 700 ABYP01000081 clade_193 N N glucuronolyticum Corynebacterium 716 FJ185225 clade_193 N N pyruviciproducens Rothia aeria 1649 DQ673320 clade_194 N N Rothia dentocariosa 1650 ADDW01000024 clade_194 N N Rothia sp. oral taxon 188 1653 GU470892 clade_194 N N Corynebacterium accolens 681 ACGD01000048 clade_195 N N Corynebacterium macginleyi 707 AB359393 clade_195 N N Corynebacterium 714 ABYQ01000237 clade_195 N N pseudogenitalium Corynebacterium 729 ACVP01000009 clade_195 N N tuberculostearicum Lactobacillus casei 1074 CP000423 clade_198 N N Lactobacillus paracasei 1106 ABQV01000067 clade_198 N N Lactobacillus zeae 1143 NR_037122 clade_198 N N Prevotella dentalis 1492 AB547678 clade_205 N N Prevotella sp. oral clone ASCG10 1529 AY923148 clade_206 N N Prevotella sp. oral clone HF050 1541 AY349399 clade_206 N N Prevotella sp. oral clone ID019 1542 AY349400 clade_206 N N Prevotella sp. oral clone IK062 1545 AY349402 clade_206 N N Prevotella genomosp. P9 oral 1499 DQ003633 clade_207 N N clone MB7_G16 Prevotella sp. oral clone AU069 1531 AY005062 clade_207 N N Prevotella sp. oral clone CY006 1532 AY005063 clade_207 N N Prevotella sp. oral clone FL019 1534 AY349392 clade_207 N N Actinomyces genomosp. C1 56 AY278610 clade_212 N N Actinomyces genomosp. C2 57 AY278611 clade_212 N N Actinomyces genomosp. P1 58 DQ003632 clade_212 N N oral clone MB6_C03 Actinomyces georgiae 59 GU561319 clade_212 N N Actinomyces israelii 60 AF479270 clade_212 N N Actinomyces massiliensis 61 AB545934 clade_212 N N Actinomyces meyeri 62 GU561321 clade_212 N N Actinomyces odontolyticus 66 ACYT01000123 clade_212 N N Actinomyces orihominis 68 AJ575186 clade_212 N N Actinomyces sp. CCUG 37290 71 AJ234058 clade_212 N N Actinomyces sp. ICM34 75 HQ616391 clade_212 N N Actinomyces sp. ICM41 76 HQ616392 clade_212 N N Actinomyces sp. ICM47 77 HQ616395 clade_212 N N Actinomyces sp. ICM54 78 HQ616398 clade_212 N N Actinomyces sp. oral clone 87 AY349366 clade_212 N N IP081 Actinomyces sp. oral taxon 91 AEUH01000060 clade_212 N N 178 Actinomyces sp. oral taxon 92 AEPP01000041 clade_212 N N 180 Actinomyces sp. TeJ5 80 GU561315 clade_212 N N Haematobacter sp. BC14248 968 GU396991 clade_213 N N Paracoccus denitrificans 1424 CP000490 clade_213 N N Paracoccus marcusii 1425 NR_044922 clade_213 N N Grimontia hollisae 967 ADAQ01000013 clade_216 N N Shewanella putrefaciens 1723 CP002457 clade_216 N N Afipia genomosp. 4 111 EU117385 clade_217 N N Rhodopseudomonas palustris 1626 CP000301 clade_217 N N Methylobacterium extorquens 1223 NC_010172 clade_218 N N Methylobacterium podarium 1224 AY468363 clade_218 N N Methylobacterium 1225 GU294320 clade_218 N N radiotolerans Methylobacterium sp. 1sub 1226 AY468371 clade_218 N N Methylobacterium sp. MM4 1227 AY468370 clade_218 N N Achromobacter denitrificans 18 NR_042021 clade_224 N N Achromobacter piechaudii 19 ADMS01000149 clade_224 N N Achromobacter xylosoxidans 20 ACRC01000072 clade_224 N N Bordetella bronchiseptica 384 NR_025949 clade_224 N OP Bordetella holmesii 385 AB683187 clade_224 N OP Bordetella parapertussis 386 NR_025950 clade_224 N OP Bordetella pertussis 387 BX640418 clade_224 N OP Microbacterium chocolatum 1230 NR_037045 clade_225 N N Microbacterium flavescens 1231 EU714363 clade_225 N N Microbacterium lacticum 1233 EU714351 clade_225 N N Microbacterium oleivorans 1234 EU714381 clade_225 N N Microbacterium oxydans 1235 EU714348 clade_225 N N Microbacterium paraoxydans 1236 AJ491806 clade_225 N N Microbacterium phyllosphaerae 1237 EU714359 clade_225 N N Microbacterium schleiferi 1238 NR_044936 clade_225 N N Microbacterium sp. 768 1239 EU714378 clade_225 N N Microbacterium sp. oral strain 1240 AF287752 clade_225 N N C24KA Microbacterium testaceum 1241 EU714365 clade_225 N N Corynebacterium atypicum 686 NR_025540 clade_229 N N Corynebacterium mastitidis 708 AB359395 clade_229 N N Corynebacterium sp. NML 725 GU238411 clade_229 N N 97_0186 Mycobacterium elephantis 1275 AF385898 clade_237 N OP Mycobacterium paraterrae 1288 EU919229 clade_237 N OP Mycobacterium phlei 1289 GU142920 clade_237 N OP Mycobacterium sp. 1776 1293 EU703152 clade_237 N N Mycobacterium sp. 1781 1294 EU703147 clade_237 N N Mycobacterium sp. AQ1GA4 1297 HM210417 clade_237 N N Mycobacterium sp. GN_10546 1299 FJ497243 clade_237 N N Mycobacterium sp. GN_10827 1300 FJ497247 clade_237 N N Mycobacterium sp. GN_11124 1301 FJ652846 clade_237 N N Mycobacterium sp. GN_9188 1302 FJ497240 clade_237 N N Mycobacterium sp. GR_2007_210 1303 FJ555538 clade_237 N N Anoxybacillus contaminans 172 NR_029006 clade_238 N N Bacillus aeolius 195 NR_025557 clade_238 N N Brevibacterium frigoritolerans 422 NR_042639 clade_238 N N Geobacillus sp. E263 934 DQ647387 clade_238 N N Geobacillus sp. WCH70 935 CP001638 clade_238 N N Geobacillus thermocatenulatus 937 NR_043020 clade_238 N N Geobacillus thermoleovorans 940 NR_074931 clade_238 N N Lysinibacillus fusiformis 1192 FN397522 clade_238 N N Planomicrobium koreense 1468 NR_025011 clade_238 N N Sporosarcina newyorkensis 1754 AFPZ01000142 clade_238 N N Sporosarcina sp. 2681 1755 GU994081 clade_238 N N Ureibacillus composti 1968 NR_043746 clade_238 N N Ureibacillus suwonensis 1969 NR_043232 clade_238 N N Ureibacillus terrenus 1970 NR_025394 clade_238 N N Ureibacillus thermophilus 1971 NR_043747 clade_238 N N Ureibacillus thermosphaericus 1972 NR_040961 clade_238 N N Prevotella micans 1507 AGWK01000061 clade_239 N N Prevotella sp. oral clone DA058 1533 AY005065 clade_239 N N Prevotella sp. SEQ053 1523 JN867222 clade_239 N N Treponema socranskii 1937 NR_024868 clade_240 N OP Treponema sp. 6:H:D15A_4 1938 AY005083 clade_240 N N Treponema sp. oral taxon 265 1953 GU408850 clade_240 N N Treponema sp. oral taxon G85 1958 GU432215 clade_240 N N Porphyromonas endodontalis 1472 ACNN01000021 clade_241 N N Porphyromonas sp. oral clone 1478 AY005068 clade_241 N N BB134 Porphyromonas sp. oral clone 1479 AY005069 clade_241 N N F016 Porphyromonas sp. oral clone 1480 AY207054 clade_241 N N P2PB_52 P1 Porphyromonas sp. oral clone 1481 AY207057 clade_241 N N P4GB_100 P2 Acidovorax sp. 98_63833 26 AY258065 clade_245 N N Comamonadaceae bacterium 663 JN585335 clade_245 N N NML000135 Comamonadaceae bacterium 664 JN585331 clade_245 N N NML790751 Comamonadaceae bacterium 665 JN585332 clade_245 N N NML910035 Comamonadaceae bacterium 666 JN585333 clade_245 N N NML910036 Comamonas sp. NSP5 668 AB076850 clade_245 N N Delftia acidovorans 748 CP000884 clade_245 N N Xenophilus aerolatus 2018 JN585329 clade_245 N N Oribacterium sp. oral taxon 1380 ACIQ02000009 clade_246 N N 078 Oribacterium sp. oral taxon 1381 GQ422713 clade_246 N N 102 Weissella cibaria 2007 NR_036924 clade_247 N N Weissella confusa 2008 NR_040816 clade_247 N N Weissella hellenica 2009 AB680902 clade_247 N N Weissella kandleri 2010 NR_044659 clade_247 N N Weissella koreensis 2011 NR_075058 clade_247 N N Weissella paramesenteroides 2012 ACKU01000017 clade_247 N N Weissella sp. KLDS 7.0701 2013 EU600924 clade_247 N N Mobiluncus curtisii 1251 AEPZ01000013 clade_249 N N Enhydrobacter aerosaccus 785 ACYI01000081 clade_256 N N Moraxella osloensis 1262 JN175341 clade_256 N N Moraxella sp. GM2 1264 JF837191 clade_256 N N Brevibacterium casei 420 JF951998 clade_257 N N Brevibacterium epidermidis 421 NR_029262 clade_257 N N Brevibacterium sanguinis 426 NR_028016 clade_257 N N Brevibacterium sp. H15 427 AB177640 clade_257 N N Acinetobacter radioresistens 35 ACVR01000010 clade_261 N N Lactobacillus alimentarius 1068 NR_044701 clade_263 N N Lactobacillus farciminis 1082 NR_044707 clade_263 N N Lactobacillus kimchii 1097 NR_025045 clade_263 N N Lactobacillus nodensis 1101 NR_041629 clade_263 N N Lactobacillus tucceti 1138 NR_042194 clade_263 N N Pseudomonas mendocina 1595 AAUL01000021 clade_265 N N Pseudomonas 1598 NR_037000 clade_265 N N pseudoalcaligenes Pseudomonas sp. NP522b 1602 EU723211 clade_265 N N Pseudomonas stutzeri 1603 AM905854 clade_265 N N Paenibacillus barcinonensis 1390 NR_042272 clade_270 N N Paenibacillus barengoltzii 1391 NR_042756 clade_270 N N Paenibacillus chibensis 1392 NR_040885 clade_270 N N Paenibacillus cookii 1393 NR_025372 clade_270 N N Paenibacillus durus 1394 NR_037017 clade_270 N N Paenibacillus glucanolyticus 1395 D78470 clade_270 N N Paenibacillus lactis 1396 NR_025739 clade_270 N N Paenibacillus pabuli 1398 NR_040853 clade_270 N N Paenibacillus popilliae 1400 NR_040888 clade_270 N N Paenibacillus sp. CIP 101062 1401 HM212646 clade_270 N N Paenibacillus sp. JC66 1404 JF824808 clade_270 N N Paenibacillus sp. R_27413 1405 HE586333 clade_270 N N Paenibacillus sp. R_27422 1406 HE586338 clade_270 N N Paenibacillus timonensis 1408 NR_042844 clade_270 N N Rothia mucilaginosa 1651 ACVO01000020 clade_271 N N Rothia nasimurium 1652 NR_025310 clade_271 N N Prevotella sp. oral taxon 302 1550 ACZK01000043 clade_280 N N Prevotella sp. oral taxon F68 1556 HM099652 clade_280 N N Prevotella tannerae 1563 ACIJ02000018 clade_280 N N Prevotellaceae bacterium 1566 AY207061 clade_280 N N P4P_62 P1 Porphyromonas 1471 AENO01000048 clade_281 N N asaccharolytica Porphyromonas gingivalis 1473 AE015924 clade_281 N N Porphyromonas macacae 1475 NR_025908 clade_281 N N Porphyromonas sp. UQD 301 1477 EU012301 clade_281 N N Porphyromonas uenonis 1482 ACLR01000152 clade_281 N N Leptotrichia buccalis 1165 CP001685 clade_282 N N Leptotrichia hofstadii 1168 ACVB02000032 clade_282 N N Leptotrichia sp. oral clone 1173 AY349386 clade_282 N N HE012 Leptotrichia sp. oral taxon 223 1176 GU408547 clade_282 N N Bacteroides fluxus 278 AFBN01000029 clade_285 N N Bacteroides helcogenes 281 CP002352 clade_285 N N Parabacteroides johnsonii 1419 ABYH01000014 clade_286 N N Parabacteroides merdae 1420 EU136685 clade_286 N N Treponema denticola 1926 ADEC01000002 clade_288 N OP Treponema genomosp. P5 oral 1929 DQ003624 clade_288 N N clone MB3_P23 Treponema putidum 1935 AJ543428 clade_288 N OP Treponema sp. oral clone 1942 AY207055 clade_288 N N P2PB_53 P3 Treponema sp. oral taxon 247 1949 GU408748 clade_288 N N Treponema sp. oral taxon 250 1950 GU408776 clade_288 N N Treponema sp. oral taxon 251 1951 GU408781 clade_288 N N Anaerococcus hydrogenalis 144 ABXA01000039 clade_289 N N Anaerococcus sp. 8404299 148 HM587318 clade_289 N N Anaerococcus sp. gpac215 156 AM176540 clade_289 N N Anaerococcus vaginalis 158 ACXU01000016 clade_289 N N Propionibacterium 1569 NC_019395 clade_290 N N acidipropionici Propionibacterium avidum 1571 AJ003055 clade_290 N N Propionibacterium granulosum 1573 FJ785716 clade_290 N N Propionibacterium jensenii 1574 NR_042269 clade_290 N N Propionibacterium propionicum 1575 NR_025277 clade_290 N N Propionibacterium sp. H456 1577 AB177643 clade_290 N N Propionibacterium thoenii 1581 NR_042270 clade_290 N N Bifidobacterium bifidum 349 ABQP01000027 clade_293 N N Leuconostoc mesenteroides 1183 ACKV01000113 clade_295 N N Leuconostoc 1184 NR_040814 clade_295 N N pseudomesenteroides Johnsonella ignava 1016 X87152 clade_298 N N Propionibacterium acnes 1570 ADJM01000010 clade_299 N N Propionibacterium sp. 1576 AFIL01000035 clade_299 N N 434_HC2 Propionibacterium sp. LG 1578 AY354921 clade_299 N N Propionibacterium sp. S555a 1579 AB264622 clade_299 N N Alicyclobacillus sp. CCUG 128 HE613268 clade_301 N N 53762 Actinomyces cardiffensis 53 GU470888 clade_303 N N Actinomyces funkei 55 HQ906497 clade_303 N N Actinomyces sp. HKU31 74 HQ335393 clade_303 N N Actinomyces sp. oral taxon 94 HM099646 clade_303 N N C55 Kerstersia gyiorum 1018 NR_025669 clade_307 N N Pigmentiphaga daeguensis 1467 JN585327 clade_307 N N Aeromonas allosaccharophila 104 S39232 clade_308 N N Aeromonas enteropelogenes 105 X71121 clade_308 N N Aeromonas hydrophila 106 NC_008570 clade_308 N N Aeromonas jandaei 107 X60413 clade_308 N N Aeromonas salmonicida 108 NC_009348 clade_308 N N Aeromonas trota 109 X60415 clade_308 N N Aeromonas veronii 110 NR_044845 clade_308 N N Marvinbryantia formatexigens 1196 AJ505973 clade_309 N N Rhodobacter sp. oral taxon 1620 HM099648 clade_310 N N C30 Rhodobacter sphaeroides 1621 CP000144 clade_310 N N Lactobacillus antri 1071 ACLL01000037 clade_313 N N Lactobacillus coleohominis 1076 ACOH01000030 clade_313 N N Lactobacillus fermentum 1083 CP002033 clade_313 N N Lactobacillus gastricus 1085 AICN01000060 clade_313 N N Lactobacillus mucosae 1099 FR693800 clade_313 N N Lactobacillus oris 1103 AEKL01000077 clade_313 N N Lactobacillus pontis 1111 HM218420 clade_313 N N Lactobacillus reuteri 1112 ACGW02000012 clade_313 N N Lactobacillus sp. KLDS 1.0707 1127 EU600911 clade_313 N N Lactobacillus sp. KLDS 1.0709 1128 EU600913 clade_313 N N Lactobacillus sp. KLDS 1.0711 1129 EU600915 clade_313 N N Lactobacillus sp. KLDS 1.0713 1131 EU600917 clade_313 N N Lactobacillus sp. KLDS 1.0716 1132 EU600921 clade_313 N N Lactobacillus sp. KLDS 1.0718 1133 EU600922 clade_313 N N Lactobacillus sp. oral taxon 1137 GQ422710 clade_313 N N 052 Lactobacillus vaginalis 1140 ACGV01000168 clade_313 N N Brevibacterium aurantiacum 419 NR_044854 clade_314 N N Brevibacterium linens 423 AJ315491 clade_314 N N Lactobacillus pentosus 1108 JN813103 clade_315 N N Lactobacillus plantarum 1110 ACGZ02000033 clade_315 N N Lactobacillus sp. KLDS 1.0702 1123 EU600906 clade_315 N N Lactobacillus sp. KLDS 1.0703 1124 EU600907 clade_315 N N Lactobacillus sp. KLDS 1.0704 1125 EU600908 clade_315 N N Lactobacillus sp. KLDS 1.0705 1126 EU600909 clade_315 N N Agrobacterium radiobacter 115 CP000628 clade_316 N N Agrobacterium tumefaciens 116 AJ389893 clade_316 N N Corynebacterium 685 EF463055 clade_317 N N argentoratense Corynebacterium diphtheriae 693 NC_002935 clade_317 N OP Corynebacterium 715 NR_037070 clade_317 N N pseudotuberculosis Corynebacterium renale 717 NR_037069 clade_317 N N Corynebacterium ulcerans 731 NR_074467 clade_317 N N Aurantimonas coralicida 191 AY065627 clade_318 N N Aureimonas altamirensis 192 FN658986 clade_318 N N Lactobacillus acidipiscis 1066 NR_024718 clade_320 N N Lactobacillus salivarius 1117 AEBA01000145 clade_320 N N Lactobacillus sp. KLDS 1.0719 1134 EU600923 clade_320 N N Lactobacillus buchneri 1073 ACGH01000101 clade_321 N N Lactobacillus genomosp. C1 1086 AY278619 clade_321 N N Lactobacillus genomosp. C2 1087 AY278620 clade_321 N N Lactobacillus hilgardii 1089 ACGP01000200 clade_321 N N Lactobacillus kefiri 1096 NR_042230 clade_321 N N Lactobacillus parabuchneri 1105 NR_041294 clade_321 N N Lactobacillus parakefiri 1107 NR_029039 clade_321 N N Lactobacillus curvatus 1079 NR_042437 clade_322 N N Lactobacillus sakei 1116 DQ989236 clade_322 N N Aneurinibacillus aneurinilyticus 167 AB101592 clade_323 N N Aneurinibacillus danicus 168 NR_028657 clade_323 N N Aneurinibacillus migulanus 169 NR_036799 clade_323 N N Aneurinibacillus terranovensis 170 NR_042271 clade_323 N N Staphylococcus aureus 1757 CP002643 clade_325 N Category-B Staphylococcus auricularis 1758 JQ624774 clade_325 N N Staphylococcus capitis 1759 ACFR01000029 clade_325 N N Staphylococcus caprae 1760 ACRH01000033 clade_325 N N Staphylococcus carnosus 1761 NR_075003 clade_325 N N Staphylococcus cohnii 1762 JN175375 clade_325 N N Staphylococcus condimenti 1763 NR_029345 clade_325 N N Staphylococcus epidermidis 1764 ACHE01000056 clade_325 N N Staphylococcus equorum 1765 NR_027520 clade_325 N N Staphylococcus haemolyticus 1767 NC_007168 clade_325 N N Staphylococcus hominis 1768 AM157418 clade_325 N N Staphylococcus lugdunensis 1769 AEQA01000024 clade_325 N N Staphylococcus pasteuri 1770 FJ189773 clade_325 N N Staphylococcus pseudintermedius 1771 CP002439 clade_325 N N Staphylococcus saccharolyticus 1772 NR_029158 clade_325 N N Staphylococcus saprophyticus 1773 NC_007350 clade_325 N N Staphylococcus sp. clone 1777 AF467424 clade_325 N N bottae7 Staphylococcus sp. H292 1775 AB177642 clade_325 N N Staphylococcus sp. H780 1776 AB177644 clade_325 N N Staphylococcus succinus 1778 NR_028667 clade_325 N N Staphylococcus warneri 1780 ACPZ01000009 clade_325 N N Staphylococcus xylosus 1781 AY395016 clade_325 N N Cardiobacterium hominis 490 ACKY01000036 clade_326 N N Cardiobacterium valvarum 491 NR_028847 clade_326 N N Pseudomonas fluorescens 1593 AY622220 clade_326 N N Pseudomonas gessardii 1594 FJ943496 clade_326 N N Pseudomonas monteilii 1596 NR_024910 clade_326 N N Pseudomonas poae 1597 GU188951 clade_326 N N Pseudomonas putida 1599 AF094741 clade_326 N N Pseudomonas sp. G1229 1601 DQ910482 clade_326 N N Pseudomonas tolaasii 1604 AF320988 clade_326 N N Pseudomonas viridiflava 1605 NR_042764 clade_326 N N Listeria grayi 1185 ACCR02000003 clade_328 N OP Listeria innocua 1186 JF967625 clade_328 N N Listeria ivanovii 1187 X56151 clade_328 N N Listeria monocytogenes 1188 CP002003 clade_328 N Category-B Listeria welshimeri 1189 AM263198 clade_328 N OP Capnocytophaga sp. oral clone 484 AY923149 clade_333 N N ASCH05 Capnocytophaga sputigena 489 ABZV01000054 clade_333 N N Leptotrichia genomosp. C1 1166 AY278621 clade_334 N N Leptotrichia shahii 1169 AY029806 clade_334 N N Leptotrichia sp. 1170 AF189244 clade_334 N N neutropenicPatient Leptotrichia sp. oral clone 1171 AY349384 clade_334 N N GT018 Leptotrichia sp. oral clone 1172 AY349385 clade_334 N N GT020 Bacteroides sp. 20_3 296 ACRQ01000064 clade_335 N N Bacteroides sp. 3_1_19 307 ADCJ01000062 clade_335 N N Bacteroides sp. 3_2_5 311 ACIB01000079 clade_335 N N Parabacteroides distasonis 1416 CP000140 clade_335 N N Parabacteroides goldsteinii 1417 AY974070 clade_335 N N Parabacteroides gordonii 1418 AB470344 clade_335 N N Parabacteroides sp. D13 1421 ACPW01000017 clade_335 N N Capnocytophaga genomosp. 477 AY278613 clade_336 N N C1 Capnocytophaga ochracea 480 AEOH01000054 clade_336 N N Capnocytophaga sp. GEJ8 481 GU561335 clade_336 N N Capnocytophaga sp. oral strain 486 AY005077 clade_336 N N A47ROY Capnocytophaga sp. S1b 482 U42009 clade_336 N N Paraprevotella clara 1426 AFFY01000068 clade_336 N N Bacteroides heparinolyticus 282 JN867284 clade_338 N N Prevotella heparinolytica 1500 GQ422742 clade_338 N N Treponema genomosp. P4 oral 1928 DQ003618 clade_339 N N clone MB2_G19 Treponema genomosp. P6 oral 1930 DQ003625 clade_339 N N clone MB4_G11 Treponema sp. oral taxon 254 1952 GU408803 clade_339 N N Treponema sp. oral taxon 508 1956 GU413616 clade_339 N N Treponema sp. oral taxon 518 1957 GU413640 clade_339 N N Chlamydia muridarum 502 AE002160 clade_341 N OP Chlamydia trachomatis 504 U68443 clade_341 N OP Chlamydia psittaci 503 NR_036864 clade_342 N Category-B Chlamydophila pneumoniae 509 NC_002179 clade_342 N OP Chlamydophila psittaci 510 D85712 clade_342 N OP Anaerococcus octavius 146 NR_026360 clade_343 N N Anaerococcus sp. 8405254 149 HM587319 clade_343 N N Anaerococcus sp. 9401487 150 HM587322 clade_343 N N Anaerococcus sp. 9403502 151 HM587325 clade_343 N N Gardnerella vaginalis 923 CP001849 clade_344 N N Campylobacter lari 466 CP000932 clade_346 N OP Anaerobiospirillum 142 NR_026075 clade_347 N N succiniciproducens Anaerobiospirillum thomasii 143 AJ420985 clade_347 N N Ruminobacter amylophilus 1654 NR_026450 clade_347 N N Succinatimonas hippei 1897 AEVO01000027 clade_347 N N Actinomyces europaeus 54 NR_026363 clade_348 N N Actinomyces sp. oral clone 82 AY349361 clade_348 N N GU009 Moraxella catarrhalis 1260 CP002005 clade_349 N N Moraxella lincolnii 1261 FR822735 clade_349 N N Moraxella sp. 16285 1263 JF682466 clade_349 N N Psychrobacter sp. 13983 1613 HM212668 clade_349 N N Actinobaculum massiliae 49 AF487679 clade_350 N N Actinobaculum schaalii 50 AY957507 clade_350 N N Actinobaculum sp. BM#101342 51 AY282578 clade_350 N N Actinobaculum sp. P2P_19 P1 52 AY207066 clade_350 N N Actinomyces sp. oral clone 84 AY349363 clade_350 N N IO076 Actinomyces sp. oral taxon 93 ACUY01000072 clade_350 N N 848 Actinomyces neuii 65 X71862 clade_352 N N Mobiluncus mulieris 1252 ACKW01000035 clade_352 N N Blastomonas natatoria 372 NR_040824 clade_356 N N Novosphingobium 1357 AAAV03000008 clade_356 N N aromaticivorans Sphingomonas sp. oral clone 1745 AY349411 clade_356 N N FI012 Sphingopyxis alaskensis 1749 CP000356 clade_356 N N Oxalobacter formigenes 1389 ACDQ01000020 clade_357 N N Veillonella atypica 1974 AEDS01000059 clade_358 N N Veillonella dispar 1975 ACIK02000021 clade_358 N N Veillonella genomosp. P1 oral 1976 DQ003631 clade_358 N N clone MB5_P17 Veillonella parvula 1978 ADFU01000009 clade_358 N N Veillonella sp. 3_1_44 1979 ADCV01000019 clade_358 N N Veillonella sp. 6_1_27 1980 ADCW01000016 clade_358 N N Veillonella sp. ACP1 1981 HQ616359 clade_358 N N Veillonella sp. AS16 1982 HQ616365 clade_358 N N Veillonella sp. BS32b 1983 HQ616368 clade_358 N N Veillonella sp. ICM51a 1984 HQ616396 clade_358 N N Veillonella sp. MSA12 1985 HQ616381 clade_358 N N Veillonella sp. NVG 100cf 1986 EF108443 clade_358 N N Veillonella sp. OK11 1987 JN695650 clade_358 N N Veillonella sp. oral clone 1990 AY923144 clade_358 N N ASCG01 Veillonella sp. oral clone 1991 AY953257 clade_358 N N ASCG02 Veillonella sp. oral clone OH1A 1992 AY947495 clade_358 N N Veillonella sp. oral taxon 158 1993 AENU01000007 clade_358 N N Kocuria marina 1040 GQ260086 clade_365 N N Kocuria rhizophila 1042 AY030315 clade_365 N N Kocuria rosea 1043 X87756 clade_365 N N Kocuria varians 1044 AF542074 clade_365 N N Clostridiaceae bacterium 531 EF451053 clade_368 N N END_2 Micrococcus antarcticus 1242 NR_025285 clade_371 N N Micrococcus luteus 1243 NR_075062 clade_371 N N Micrococcus lylae 1244 NR_026200 clade_371 N N Micrococcus sp. 185 1245 EU714334 clade_371 N N Lactobacillus brevis 1072 EU194349 clade_372 N N Lactobacillus parabrevis 1104 NR_042456 clade_372 N N Pediococcus acidilactici 1436 ACXB01000026 clade_372 N N Pediococcus pentosaceus 1437 NR_075052 clade_372 N N Lactobacillus dextrinicus 1081 NR_036861 clade_373 N N Lactobacillus perolens 1109 NR_029360 clade_373 N N Lactobacillus rhamnosus 1113 ABWJ01000068 clade_373 N N Lactobacillus saniviri 1118 AB602569 clade_373 N N Lactobacillus sp. BT6 1121 HQ616370 clade_373 N N Mycobacterium mageritense 1282 FR798914 clade_374 N OP Mycobacterium neoaurum 1286 AF268445 clade_374 N OP Mycobacterium smegmatis 1291 CP000480 clade_374 N OP Mycobacterium sp. HE5 1304 AJ012738 clade_374 N N Dysgonomonas gadei 775 ADLV01000001 clade_377 N N Dysgonomonas mossii 776 ADLW01000023 clade_377 N N Porphyromonas levii 1474 NR_025907 clade_377 N N Porphyromonas somerae 1476 AB547667 clade_377 N N Bacteroides barnesiae 267 NR_041446 clade_378 N N Bacteroides coprocola 272 ABIY02000050 clade_378 N N Bacteroides coprophilus 273 ACBW01000012 clade_378 N N Bacteroides dorei 274 ABWZ01000093 clade_378 N N Bacteroides massiliensis 284 AB200226 clade_378 N N Bacteroides plebeius 289 AB200218 clade_378 N N Bacteroides sp. 3_1_33FAA 309 ACPS01000085 clade_378 N N Bacteroides sp. 3_1_40A 310 ACRT01000136 clade_378 N N Bacteroides sp. 4_3_47FAA 313 ACDR02000029 clade_378 N N Bacteroides sp. 9_1_42FAA 314 ACAA01000096 clade_378 N N Bacteroides sp. NB_8 323 AB117565 clade_378 N N Bacteroides vulgatus 331 CP000139 clade_378 N N Bacteroides ovatus 287 ACWH01000036 clade_38 N N Bacteroides sp. 1_1_30 294 ADCL01000128 clade_38 N N Bacteroides sp. 2_1_22 297 ACPQ01000117 clade_38 N N Bacteroides sp. 2_2_4 299 ABZZ01000168 clade_38 N N Bacteroides sp. 3_1_23 308 ACRS01000081 clade_38 N N Bacteroides sp. D1 318 ACAB02000030 clade_38 N N Bacteroides sp. D2 321 ACGA01000077 clade_38 N N Bacteroides sp. D22 320 ADCK01000151 clade_38 N N Bacteroides xylanisolvens 332 ADKP01000087 clade_38 N N Treponema lecithinolyticum 1931 NR_026247 clade_380 N OP Treponema parvum 1933 AF302937 clade_380 N OP Treponema sp. oral clone 1940 AY349417 clade_380 N N JU025 Treponema sp. oral taxon 270 1954 GQ422733 clade_380 N N Parascardovia denticolens 1428 ADEB01000020 clade_381 N N Scardovia inopinata 1688 AB029087 clade_381 N N Scardovia wiggsiae 1689 AY278626 clade_381 N N Clostridiales bacterium 533 HM587320 clade_384 N N 9400853 Mogibacterium diversum 1254 NR_027191 clade_384 N N Mogibacterium neglectum 1255 NR_027203 clade_384 N N Mogibacterium pumilum 1256 NR_028608 clade_384 N N Mogibacterium timidum 1257 Z36296 clade_384 N N Borrelia burgdorferi 389 ABGI01000001 clade_386 N OP Borrelia garinii 392 ABJV01000001 clade_386 N OP Borrelia sp. NE49 397 AJ224142 clade_386 N OP Caldimonas manganoxidans 457 NR_040787 clade_387 N N Comamonadaceae bacterium 667 HM099651 clade_387 N N oral taxon F47 Lautropia mirabilis 1149 AEQP01000026 clade_387 N N Lautropia sp. oral clone AP009 1150 AY005030 clade_387 N N Peptoniphilus asaccharolyticus 1441 D14145 clade_389 N N Peptoniphilus duerdenii 1442 EU526290 clade_389 N N Peptoniphilus harei 1443 NR_026358 clade_389 N N Peptoniphilus indolicus 1444 AY153431 clade_389 N N Peptoniphilus lacrimalis 1446 ADDO01000050 clade_389 N N Peptoniphilus sp. gpac077 1450 AM176527 clade_389 N N Peptoniphilus sp. JC140 1447 JF824803 clade_389 N N Peptoniphilus sp. oral taxon 1452 ADCS01000031 clade_389 N N 386 Peptoniphilus sp. oral taxon 1453 AEAA01000090 clade_389 N N 836 Peptostreptococcaceae 1454 JN837495 clade_389 N N bacterium ph1 Dialister pneumosintes 765 HM596297 clade_390 N N Dialister sp. oral taxon 502 767 GQ422739 clade_390 N N Cupriavidus metallidurans 741 GU230889 clade_391 N N Herbaspirillum seropedicae 1001 CP002039 clade_391 N N Herbaspirillum sp. JC206 1002 JN657219 clade_391 N N Janthinobacterium sp. SY12 1015 EF455530 clade_391 N N Massilia sp. CCUG 43427A 1197 FR773700 clade_391 N N Ralstonia pickettii 1615 NC_010682 clade_391 N N Ralstonia sp. 5_7_47FAA 1616 ACUF01000076 clade_391 N N Francisella novicida 889 ABSS01000002 clade_392 N N Francisella philomiragia 890 AY928394 clade_392 N N Francisella tularensis 891 ABAZ01000082 clade_392 N Category-A Ignatzschineria indica 1009 HQ823562 clade_392 N N Ignatzschineria sp. NML 1010 HQ823559 clade_392 N N 95_0260 Streptococcus mutans 1814 AP010655 clade_394 N N Lactobacillus gasseri 1084 ACOZ01000018 clade_398 N N Lactobacillus hominis 1090 FR681902 clade_398 N N Lactobacillus iners 1091 AEKJ01000002 clade_398 N N Lactobacillus johnsonii 1093 AE017198 clade_398 N N Lactobacillus senioris 1119 AB602570 clade_398 N N Lactobacillus sp. oral clone 1135 AY349382 clade_398 N N HT002 Weissella beninensis 2006 EU439435 clade_398 N N Sphingomonas echinoides 1744 NR_024700 clade_399 N N Sphingomonas sp. oral taxon 1747 HM099639 clade_399 N N A09 Sphingomonas sp. oral taxon 1748 HM099645 clade_399 N N F71 Zymomonas mobilis 2032 NR_074274 clade_399 N N Arcanobacterium 174 NR_025347 clade_400 N N haemolyticum Arcanobacterium pyogenes 175 GU585578 clade_400 N N Trueperella pyogenes 1962 NR_044858 clade_400 N N Lactococcus garvieae 1144 AF061005 clade_401 N N Lactococcus lactis 1145 CP002365 clade_401 N N Brevibacterium mcbrellneri 424 ADNU01000076 clade_402 N N Brevibacterium paucivorans 425 EU086796 clade_402 N N Brevibacterium sp. JC43 428 JF824806 clade_402 N N Selenomonas artemidis 1692 HM596274 clade_403 N N Selenomonas sp. FOBRC9 1704 HQ616378 clade_403 N N Selenomonas sp. oral taxon 1715 AENV01000007 clade_403 N N 137 Desmospora activa 751 AM940019 clade_404 N N Desmospora sp. 8437 752 AFHT01000143 clade_404 N N Paenibacillus sp. oral taxon 1407 HM099647 clade_404 N N F45 Corynebacterium 682 ADNS01000011 clade_405 N N ammoniagenes Corynebacterium 687 ACLH01000041 clade_405 N N aurimucosum Corynebacterium bovis 688 AF537590 clade_405 N N Corynebacterium canis 689 GQ871934 clade_405 N N Corynebacterium casei 690 NR_025101 clade_405 N N Corynebacterium durum 694 Z97069 clade_405 N N Corynebacterium efficiens 695 ACLI01000121 clade_405 N N Corynebacterium falsenii 696 Y13024 clade_405 N N Corynebacterium flavescens 697 NR_037040 clade_405 N N Corynebacterium glutamicum 701 BA000036 clade_405 N N Corynebacterium jeikeium 704 ACYW01000001 clade_405 N OP Corynebacterium 705 NR_026380 clade_405 N N kroppenstedtii Corynebacterium 706 ACHJ01000075 clade_405 N N lipophiloflavum Corynebacterium matruchotii 709 ACSH02000003 clade_405 N N Corynebacterium 710 X82064 clade_405 N N minutissimum Corynebacterium resistens 718 ADGN01000058 clade_405 N N Corynebacterium simulans 720 AF537604 clade_405 N N Corynebacterium singulare 721 NR_026394 clade_405 N N Corynebacterium sp. 1 ex 722 Y13427 clade_405 N N sheep Corynebacterium sp. NML 726 GU238413 clade_405 N N 99_0018 Corynebacterium striatum 727 ACGE01000001 clade_405 N OP Corynebacterium urealyticum 732 X81913 clade_405 N OP Corynebacterium variabile 734 NR_025314 clade_405 N N Aerococcus sanguinicola 98 AY837833 clade_407 N N Aerococcus urinae 99 CP002512 clade_407 N N Aerococcus urinaeequi 100 NR_043443 clade_407 N N Aerococcus viridans 101 ADNT01000041 clade_407 N N Fusobacterium naviforme 898 HQ223106 clade_408 N N Moryella indoligenes 1268 AF527773 clade_408 N N Selenomonas genomosp. P5 1697 AY341820 clade_410 N N Selenomonas sp. oral clone 1710 AY349408 clade_410 N N IQ048 Selenomonas sputigena 1717 ACKP02000033 clade_410 N N Hyphomicrobium sulfonivorans 1007 AY468372 clade_411 N N Methylocella silvestris 1228 NR_074237 clade_411 N N Legionella pneumophila 1153 NC_002942 clade_412 N OP Lactobacillus coryniformis 1077 NR_044705 clade_413 N N Arthrobacter agilis 178 NR_026198 clade_414 N N Arthrobacter arilaitensis 179 NR_074608 clade_414 N N Arthrobacter bergerei 180 NR_025612 clade_414 N N Arthrobacter globiformis 181 NR_026187 clade_414 N N Arthrobacter nicotianae 182 NR_026190 clade_414 N N Mycobacterium abscessus 1269 AGQU01000002 clade_418 N OP Mycobacterium chelonae 1273 AB548610 clade_418 N OP Bacteroides salanitronis 291 CP002530 clade_419 N N Paraprevotella xylaniphila 1427 AFBR01000011 clade_419 N N Barnesiella intestinihominis 336 AB370251 clade_420 N N Barnesiella viscericola 337 NR_041508 clade_420 N N Parabacteroides sp. NS31_3 1422 JN029805 clade_420 N N Porphyromonadaceae 1470 EF184292 clade_420 N N bacterium NML 060648 Tannerella forsythia 1913 CP003191 clade_420 N N Tannerella sp. 1914 ACWX01000068 clade_420 N N 6_1_58FAA_CT1 Mycoplasma amphoriforme 1311 AY531656 clade_421 N N Mycoplasma genitalium 1317 L43967 clade_421 N N Mycoplasma pneumoniae 1322 NC_000912 clade_421 N N Mycoplasma penetrans 1321 NC_004432 clade_422 N N Ureaplasma parvum 1966 AE002127 clade_422 N N Ureaplasma urealyticum 1967 AAYN01000002 clade_422 N N Treponema genomosp. P1 1927 AY341822 clade_425 N N Treponema sp. oral taxon 228 1943 GU408580 clade_425 N N Treponema sp. oral taxon 230 1944 GU408603 clade_425 N N Treponema sp. oral taxon 231 1945 GU408631 clade_425 N N Treponema sp. oral taxon 232 1946 GU408646 clade_425 N N Treponema sp. oral taxon 235 1947 GU408673 clade_425 N N Treponema sp. ovine footrot 1959 AJ010951 clade_425 N N Treponema vincentii 1960 ACYH01000036 clade_425 N OP Burkholderiales bacterium 452 ADCQ01000066 clade_432 N OP 1_1_47 Parasutterella 1429 AFBP01000029 clade_432 N N excrementihominis Parasutterella secunda 1430 AB491209 clade_432 N N Sutterella morbirenis 1898 AJ832129 clade_432 N N Sutterella sanguinus 1900 AJ748647 clade_432 N N Sutterella sp. YIT 12072 1901 AB491210 clade_432 N N Sutterella stercoricanis 1902 NR_025600 clade_432 N N Sutterella wadsworthensis 1903 ADMF01000048 clade_432 N N Propionibacterium 1572 NR_036972 clade_433 N N freudenreichii Propionibacterium sp. oral 1580 GQ422728 clade_433 N N taxon 192 Tessaracoccus sp. oral taxon 1917 HM099640 clade_433 N N F04 Peptoniphilus ivorii 1445 Y07840 clade_434 N N Peptoniphilus sp. gpac007 1448 AM176517 clade_434 N N Peptoniphilus sp. gpac018A 1449 AM176519 clade_434 N N Peptoniphilus sp. gpac148 1451 AM176535 clade_434 N N Flexispira rappini 887 AY126479 clade_436 N N Helicobacter bilis 993 ACDN01000023 clade_436 N N Helicobacter cinaedi 995 ABQT01000054 clade_436 N N Helicobacter sp. None 998 U44756 clade_436 N N Brevundimonas subvibrioides 429 CP002102 clade_438 N N Hyphomonas neptunium 1008 NR_074092 clade_438 N N Phenylobacterium zucineum 1465 AY628697 clade_438 N N Streptococcus downei 1793 AEKN01000002 clade_441 N N Streptococcus sp. SHV515 1848 Y07601 clade_441 N N Acinetobacter sp. CIP 53.82 40 JQ638584 clade_443 N N Halomonas elongata 990 NR_074782 clade_443 N N Halomonas johnsoniae 991 FR775979 clade_443 N N Butyrivibrio fibrisolvens 456 U41172 clade_444 N N Roseburia sp. 11SE37 1640 FM954975 clade_444 N N Roseburia sp. 11SE38 1641 FM954976 clade_444 N N Shuttleworthia satelles 1728 ACIP02000004 clade_444 N N Shuttleworthia sp. MSX8B 1729 HQ616383 clade_444 N N Shuttleworthia sp. oral taxon 1730 GU432167 clade_444 N N G69 Bdellovibrio sp. MPA 344 AY294215 clade_445 N N Desulfobulbus sp. oral clone 755 AY005036 clade_445 N N CH031 Desulfovibrio desulfuricans 757 DQ092636 clade_445 N N Desulfovibrio fairfieldensis 758 U42221 clade_445 N N Desulfovibrio piger 759 AF192152 clade_445 N N Desulfovibrio sp. 3_1_syn3 760 ADDR01000239 clade_445 N N Geobacter bemidjiensis 941 CP001124 clade_445 N N Brachybacterium alimentarium 401 NR_026269 clade_446 N N Brachybacterium 402 AB537169 clade_446 N N conglomeratum Brachybacterium 403 NR_026272 clade_446 N N tyrofermentans Dermabacter hominis 749 FJ263375 clade_446 N N Aneurinibacillus 171 NR_029303 clade_448 N N thermoaerophilus Brevibacillus agri 409 NR_040983 clade_448 N N Brevibacillus centrosporus 411 NR_043414 clade_448 N N Brevibacillus choshinensis 412 NR_040980 clade_448 N N Brevibacillus invocatus 413 NR_041836 clade_448 N N Brevibacillus parabrevis 415 NR_040981 clade_448 N N Brevibacillus reuszeri 416 NR_040982 clade_448 N N Brevibacillus sp. phR 417 JN837488 clade_448 N N Brevibacillus thermoruber 418 NR_026514 clade_448 N N Lactobacillus murinus 1100 NR_042231 clade_449 N N Lactobacillus oeni 1102 NR_043095 clade_449 N N Lactobacillus ruminis 1115 ACGS02000043 clade_449 N N Lactobacillus vini 1141 NR_042196 clade_449 N N Gemella haemolysans 924 ACDZ02000012 clade_450 N N Gemella morbillorum 925 NR_025904 clade_450 N N Gemella morbillorum 926 ACRX01000010 clade_450 N N Gemella sanguinis 927 ACRY01000057 clade_450 N N Gemella sp. oral clone 929 AY923133 clade_450 N N ASCE02 Gemella sp. oral clone 930 AY923139 clade_450 N N ASCF04 Gemella sp. oral clone 931 AY923143 clade_450 N N ASCF12 Gemella sp. WAL 1945J 928 EU427463 clade_450 N N Sporolactobacillus nakayamae 1753 NR_042247 clade_451 N N Gluconacetobacter entanii 945 NR_028909 clade_452 N N Gluconacetobacter europaeus 946 NR_026513 clade_452 N N Gluconacetobacter hansenii 947 NR_026133 clade_452 N N Gluconacetobacter oboediens 949 NR_041295 clade_452 N N Gluconacetobacter xylinus 950 NR_074338 clade_452 N N Auritibacter ignavus 193 FN554542 clade_453 N N Dermacoccus sp. Ellin185 750 AEIQ01000090 clade_453 N N Janibacter limosus 1013 NR_026362 clade_453 N N Janibacter melonis 1014 EF063716 clade_453 N N Acetobacter aceti 7 NR_026121 clade_454 N N Acetobacter fabarum 8 NR_042678 clade_454 N N Acetobacter lovaniensis 9 NR_040832 clade_454 N N Acetobacter malorum 10 NR_025513 clade_454 N N Acetobacter orientalis 11 NR_028625 clade_454 N N Acetobacter pasteurianus 12 NR_026107 clade_454 N N Acetobacter pomorum 13 NR_042112 clade_454 N N Acetobacter syzygii 14 NR_040868 clade_454 N N Acetobacter tropicalis 15 NR_036881 clade_454 N N Gluconacetobacter 943 NR_028767 clade_454 N N azotocaptans Gluconacetobacter 944 NR_074292 clade_454 N N diazotrophicus Gluconacetobacter johannae 948 NR_024959 clade_454 N N Nocardia brasiliensis 1351 AIHV01000038 clade_455 N N Nocardia cyriacigeorgica 1352 HQ009486 clade_455 N N Nocardia puris 1354 NR_028994 clade_455 N N Nocardia sp. 01_Je_025 1355 GU574059 clade_455 N N Rhodococcus equi 1623 ADNW01000058 clade_455 N N Oceanobacillus caeni 1358 NR_041533 clade_456 N N Oceanobacillus sp. Ndiop 1359 CAER01000083 clade_456 N N Ornithinibacillus bavariensis 1384 NR_044923 clade_456 N N Ornithinibacillus sp. 7_10AIA 1385 FN397526 clade_456 N N Virgibacillus proomii 2005 NR_025308 clade_456 N N Corynebacterium amycolatum 683 ABZU01000033 clade_457 N OP Corynebacterium hansenii 702 AM946639 clade_457 N N Corynebacterium xerosis 735 FN179330 clade_457 N OP Staphylococcaceae bacterium 1756 AY841362 clade_458 N N NML 92_0017 Staphylococcus fleurettii 1766 NR_041326 clade_458 N N Staphylococcus sciuri 1774 NR_025520 clade_458 N N Staphylococcus vitulinus 1779 NR_024670 clade_458 N N Stenotrophomonas maltophilia 1782 AAVZ01000005 clade_459 N N Stenotrophomonas sp. FG_6 1783 EF017810 clade_459 N N Mycobacterium africanum 1270 AF480605 clade_46 N OP Mycobacterium alsiensis 1271 AJ938169 clade_46 N OP Mycobacterium avium 1272 CP000479 clade_46 N OP Mycobacterium colombiense 1274 AM062764 clade_46 N OP Mycobacterium gordonae 1276 GU142930 clade_46 N OP Mycobacterium intracellulare 1277 GQ153276 clade_46 N OP Mycobacterium kansasii 1278 AF480601 clade_46 N OP Mycobacterium lacus 1279 NR_025175 clade_46 N OP Mycobacterium leprae 1280 FM211192 clade_46 N OP Mycobacterium lepromatosis 1281 EU203590 clade_46 N OP Mycobacterium mantenii 1283 FJ042897 clade_46 N OP Mycobacterium marinum 1284 NC_010612 clade_46 N OP Mycobacterium microti 1285 NR_025234 clade_46 N OP Mycobacterium 1287 ADNV01000350 clade_46 N OP parascrofulaceum Mycobacterium seoulense 1290 DQ536403 clade_46 N OP Mycobacterium sp. 1761 1292 EU703150 clade_46 N N Mycobacterium sp. 1791 1295 EU703148 clade_46 N N Mycobacterium sp. 1797 1296 EU703149 clade_46 N N Mycobacterium sp. 1298 HQ174245 clade_46 N N B10_07.09.0206 Mycobacterium sp. 1305 HM627011 clade_46 N N NLA001000736 Mycobacterium sp. W 1306 DQ437715 clade_46 N N Mycobacterium tuberculosis 1307 CP001658 clade_46 N Category-C Mycobacterium ulcerans 1308 AB548725 clade_46 N OP Mycobacterium vulneris 1309 EU834055 clade_46 N OP Xanthomonas campestris 2016 EF101975 clade_461 N N Xanthomonas sp. kmd_489 2017 EU723184 clade_461 N N Dietzia natronolimnaea 769 GQ870426 clade_462 N N Dietzia sp. BBDP51 770 DQ337512 clade_462 N N Dietzia sp. CA149 771 GQ870422 clade_462 N N Dietzia timorensis 772 GQ870424 clade_462 N N Gordonia bronchialis 951 NR_027594 clade_463 N N Gordonia polyisoprenivorans 952 DQ385609 clade_463 N N Gordonia sp. KTR9 953 DQ068383 clade_463 N N Gordonia sputi 954 FJ536304 clade_463 N N Gordonia terrae 955 GQ848239 clade_463 N N Leptotrichia goodfellowii 1167 ADAD01000110 clade_465 N N Leptotrichia sp. oral clone 1174 AY349387 clade_465 N N IK040 Leptotrichia sp. oral clone 1175 AY207053 clade_465 N N P2PB_51 P1 Bacteroidales genomosp. P7 264 DQ003623 clade_466 N N oral clone MB3_P19 Butyricimonas virosa 454 AB443949 clade_466 N N Odoribacter laneus 1363 AB490805 clade_466 N N Odoribacter splanchnicus 1364 CP002544 clade_466 N N Capnocytophaga gingivalis 478 ACLQ01000011 clade_467 N N Capnocytophaga granulosa 479 X97248 clade_467 N N Capnocytophaga sp. oral clone 483 AY005074 clade_467 N N AH015 Capnocytophaga sp. oral strain 487 AY005073 clade_467 N N S3 Capnocytophaga sp. oral taxon 488 AEXX01000050 clade_467 N N 338 Capnocytophaga canimorsus 476 CP002113 clade_468 N N Capnocytophaga sp. oral clone 485 AY349368 clade_468 N N ID062 Lactobacillus catenaformis 1075 M23729 clade_469 N N Lactobacillus vitulinus 1142 NR_041305 clade_469 N N Cetobacterium somerae 501 AJ438155 clade_470 N N Fusobacterium gonidiaformans 896 ACET01000043 clade_470 N N Fusobacterium mortiferum 897 ACDB02000034 clade_470 N N Fusobacterium necrogenes 899 X55408 clade_470 N N Fusobacterium necrophorum 900 AM905356 clade_470 N N Fusobacterium sp. 12_1B 905 AGWJ01000070 clade_470 N N Fusobacterium sp. 3_1_5R 911 ACDD01000078 clade_470 N N Fusobacterium sp. D12 918 ACDG02000036 clade_470 N N Fusobacterium ulcerans 921 ACDH01000090 clade_470 N N Fusobacterium varium 922 ACIE01000009 clade_470 N N Mycoplasma arthritidis 1312 NC_011025 clade_473 N N Mycoplasma faucium 1314 NR_024983 clade_473 N N Mycoplasma hominis 1318 AF443616 clade_473 N N Mycoplasma orale 1319 AY796060 clade_473 N N Mycoplasma salivarium 1324 M24661 clade_473 N N Mitsuokella jalaludinii 1247 NR_028840 clade_474 N N Mitsuokella multacida 1248 ABWK02000005 clade_474 N N Mitsuokella sp. oral taxon 521 1249 GU413658 clade_474 N N Mitsuokella sp. oral taxon G68 1250 GU432166 clade_474 N N Selenomonas genomosp. C1 1695 AY278627 clade_474 N N Selenomonas genomosp. P8 1700 DQ003628 clade_474 N N oral clone MB5_P06 Selenomonas ruminantium 1703 NR_075026 clade_474 N N Veillonellaceae bacterium oral 1994 GU402916 clade_474 N N taxon 131 Alloscardovia omnicolens 139 NR_042583 clade_475 N N Alloscardovia sp. OB7196 140 AB425070 clade_475 N N Bifidobacterium urinalis 366 AJ278695 clade_475 N N Prevotella loescheii 1503 JN867231 clade_48 N N Prevotella sp. oral clone 1530 DQ272511 clade_48 N N ASCG12 Prevotella sp. oral clone 1540 AY349398 clade_48 N N GU027 Prevotella sp. oral taxon 472 1553 ACZS01000106 clade_48 N N Selenomonas dianae 1693 GQ422719 clade_480 N N Selenomonas flueggei 1694 AF287803 clade_480 N N Selenomonas genomosp. C2 1696 AY278628 clade_480 N N Selenomonas genomosp. P6 1698 DQ003636 clade_480 N N oral clone MB3_C41 Selenomonas genomosp. P7 1699 DQ003627 clade_480 N N oral clone MB5_C08 Selenomonas infelix 1701 AF287802 clade_480 N N Selenomonas noxia 1702 GU470909 clade_480 N N Selenomonas sp. oral clone 1705 AY349403 clade_480 N N FT050 Selenomonas sp. oral clone 1706 AY349404 clade_480 N N GI064 Selenomonas sp. oral clone 1707 AY349405 clade_480 N N GT010 Selenomonas sp. oral clone 1708 AY349406 clade_480 N N HU051 Selenomonas sp. oral clone 1709 AY349407 clade_480 N N IK004 Selenomonas sp. oral clone 1711 AY349409 clade_480 N N JI021 Selenomonas sp. oral clone 1712 AY349410 clade_480 N N JS031 Selenomonas sp. oral clone 1713 AY947498 clade_480 N N OH4A Selenomonas sp. oral clone 1714 AY207052 clade_480 N N P2PA_80 P4 Selenomonas sp. oral taxon 1716 AEEJ01000007 clade_480 N N 149 Veillonellaceae bacterium oral 1995 GU470897 clade_480 N N taxon 155 Agrococcus jenensis 117 NR_026275 clade_484 N N Microbacterium gubbeenense 1232 NR_025098 clade_484 N N Pseudoclavibacter sp. Timone 1590 FJ375951 clade_484 N N Tropheryma whipplei 1961 BX251412 clade_484 N N Zimmermannella bifida 2031 AB012592 clade_484 N N Legionella hackeliae 1151 M36028 clade_486 N OP Legionella longbeachae 1152 M36029 clade_486 N OP Legionella sp. D3923 1154 JN380999 clade_486 N OP Legionella sp. D4088 1155 JN381012 clade_486 N OP Legionella sp. H63 1156 JF831047 clade_486 N OP Legionella sp. NML 93L054 1157 GU062706 clade_486 N OP Legionella steelei 1158 HQ398202 clade_486 N OP Tatlockia micdadei 1915 M36032 clade_486 N N Helicobacter pullorum 996 ABQU01000097 clade_489 N N Acetobacteraceae bacterium 16 AGEZ01000040 clade_490 N N AT_5844 Roseomonas cervicalis 1643 ADVL01000363 clade_490 N N Roseomonas mucosa 1644 NR_028857 clade_490 N N Roseomonas sp. NML94_0193 1645 AF533357 clade_490 N N Roseomonas sp. NML97_0121 1646 AF533359 clade_490 N N Roseomonas sp. NML98_0009 1647 AF533358 clade_490 N N Roseomonas sp. NML98_0157 1648 AF533360 clade_490 N N Rickettsia akari 1627 CP000847 clade_492 N OP Rickettsia conorii 1628 AE008647 clade_492 N OP Rickettsia prowazekii 1629 M21789 clade_492 N Category-B Rickettsia rickettsii 1630 NC_010263 clade_492 N OP Rickettsia slovaca 1631 L36224 clade_492 N OP Rickettsia typhi 1632 AE017197 clade_492 N OP Anaeroglobus geminatus 160 AGCJ01000054 clade_493 N N Megasphaera genomosp. C1 1201 AY278622 clade_493 N N Megasphaera micronuciformis 1203 AECS01000020 clade_493 N N Clostridiales genomosp. 540 CP001850 clade_495 N N BVAB3 Tsukamurella paurometabola 1963 X80628 clade_496 N N Tsukamurella tyrosinosolvens 1964 AB478958 clade_496 N N Abiotrophia para_adiacens 2 AB022027 clade_497 N N Carnobacterium divergens 492 NR_044706 clade_497 N N Carnobacterium 493 NC_019425 clade_497 N N maltaromaticum Enterococcus avium 800 AF133535 clade_497 N N Enterococcus caccae 801 AY943820 clade_497 N N Enterococcus casseliflavus 802 AEWT01000047 clade_497 N N Enterococcus durans 803 AJ276354 clade_497 N N Enterococcus faecalis 804 AE016830 clade_497 N N Enterococcus faecium 805 AM157434 clade_497 N N Enterococcus gallinarum 806 AB269767 clade_497 N N Enterococcus gilvus 807 AY033814 clade_497 N N Enterococcus hawaiiensis 808 AY321377 clade_497 N N Enterococcus hirae 809 AF061011 clade_497 N N Enterococcus italicus 810 AEPV01000109 clade_497 N N Enterococcus mundtii 811 NR_024906 clade_497 N N Enterococcus raffinosus 812 FN600541 clade_497 N N Enterococcus sp. BV2CASA2 813 JN809766 clade_497 N N Enterococcus sp. CCRI_16620 814 GU457263 clade_497 N N Enterococcus sp. F95 815 FJ463817 clade_497 N N Enterococcus sp. RfL6 816 AJ133478 clade_497 N N Enterococcus thailandicus 817 AY321376 clade_497 N N Fusobacterium canifelinum 893 AY162222 clade_497 N N Fusobacterium genomosp. C1 894 AY278616 clade_497 N N Fusobacterium genomosp. C2 895 AY278617 clade_497 N N Fusobacterium periodonticum 902 ACJY01000002 clade_497 N N Fusobacterium sp. 1_1_41FAA 906 ADGG01000053 clade_497 N N Fusobacterium sp. 11_3_2 904 ACUO01000052 clade_497 N N Fusobacterium sp. 2_1_31 907 ACDC02000018 clade_497 N N Fusobacterium sp. 3_1_27 908 ADGF01000045 clade_497 N N Fusobacterium sp. 3_1_33 909 ACQE01000178 clade_497 N N Fusobacterium sp. 3_1_36A2 910 ACPU01000044 clade_497 N N Fusobacterium sp. AC18 912 HQ616357 clade_497 N N Fusobacterium sp. ACB2 913 HQ616358 clade_497 N N Fusobacterium sp. AS2 914 HQ616361 clade_497 N N Fusobacterium sp. CM1 915 HQ616371 clade_497 N N Fusobacterium sp. CM21 916 HQ616375 clade_497 N N Fusobacterium sp. CM22 917 HQ616376 clade_497 N N Fusobacterium sp. oral clone 919 AY923141 clade_497 N N ASCF06 Fusobacterium sp. oral clone 920 AY953256 clade_497 N N ASCF11 Granulicatella adiacens 959 ACKZ01000002 clade_497 N N Granulicatella elegans 960 AB252689 clade_497 N N Granulicatella paradiacens 961 AY879298 clade_497 N N Granulicatella sp. oral clone 963 AY923126 clade_497 N N ASC02 Granulicatella sp. oral clone 964 DQ341469 clade_497 N N ASCA05 Granulicatella sp. oral clone 965 AY953251 clade_497 N N ASCB09 Granulicatella sp. oral clone 966 AY923146 clade_497 N N ASCG05 Tetragenococcus halophilus 1918 NR_075020 clade_497 N N Tetragenococcus koreensis 1919 NR_043113 clade_497 N N Vagococcus fluvialis 1973 NR_026489 clade_497 N N Chryseobacterium anthropi 514 AM982793 clade_498 N N Chryseobacterium gleum 515 ACKQ02000003 clade_498 N N Chryseobacterium hominis 516 NR_042517 clade_498 N N Treponema refringens 1936 AF426101 clade_499 N OP Treponema sp. oral clone 1941 AY349416 clade_499 N N JU031 Treponema sp. oral taxon 239 1948 GU408738 clade_499 N N Treponema sp. oral taxon 271 1955 GU408871 clade_499 N N Alistipes finegoldii 129 NR_043064 clade_500 N N Alistipes onderdonkii 131 NR_043318 clade_500 N N Alistipes putredinis 132 ABFK02000017 clade_500 N N Alistipes shahii 133 FP929032 clade_500 N N Alistipes sp. HGB5 134 AENZ01000082 clade_500 N N Alistipes sp. JC50 135 JF824804 clade_500 N N Alistipes sp. RMA 9912 136 GQ140629 clade_500 N N Mycoplasma agalactiae 1310 AF010477 clade_501 N N Mycoplasma bovoculi 1313 NR_025987 clade_501 N N Mycoplasma fermentans 1315 CP002458 clade_501 N N Mycoplasma flocculare 1316 X62699 clade_501 N N Mycoplasma ovipneumoniae 1320 NR_025989 clade_501 N N Arcobacter butzleri 176 AEPT01000071 clade_502 N N Arcobacter cryaerophilus 177 NR_025905 clade_502 N N Campylobacter curvus 461 NC_009715 clade_502 N OP Campylobacter rectus 467 ACFU01000050 clade_502 N OP Campylobacter showae 468 ACVQ01000030 clade_502 N OP Campylobacter sp. FOBRC14 469 HQ616379 clade_502 N OP Campylobacter sp. FOBRC15 470 HQ616380 clade_502 N OP Campylobacter sp. oral clone 471 AY005038 clade_502 N OP BB120 Campylobacter sputorum 472 NR_044839 clade_502 N OP Bacteroides ureolyticus 330 GQ167666 clade_504 N N Campylobacter gracilis 463 ACYG01000026 clade_504 N OP Campylobacter hominis 464 NC_009714 clade_504 N OP Dialister invisus 762 ACIM02000001 clade_506 N N Dialister micraerophilus 763 AFBB01000028 clade_506 N N Dialister microaerophilus 764 AENT01000008 clade_506 N N Dialister propionicifaciens 766 NR_043231 clade_506 N N Dialister succinatiphilus 768 AB370249 clade_506 N N Megasphaera elsdenii 1200 AY038996 clade_506 N N Megasphaera genomosp. 1202 ADGP01000010 clade_506 N N type_1 Megasphaera sp. BLPYG_07 1204 HM990964 clade_506 N N Megasphaera sp. UPII 199_6 1205 AFIJ01000040 clade_506 N N Chromobacterium violaceum 513 NC_005085 clade_507 N N Laribacter hongkongensis 1148 CP001154 clade_507 N N Methylophilus sp. ECd5 1229 AY436794 clade_507 N N Finegoldia magna 883 ACHM02000001 clade_509 N N Parvimonas micra 1431 AB729072 clade_509 N N Parvimonas sp. oral taxon 110 1432 AFII01000002 clade_509 N N Peptostreptococcus micros 1456 AM176538 clade_509 N N Peptostreptococcus sp. oral 1460 AY349390 clade_509 N N clone FJ023 Peptostreptococcus sp. 1458 AY207059 clade_509 N N P4P_31 P3 Helicobacter pylori 997 CP000012 clade_510 N OP Anaplasma marginale 165 ABOR01000019 clade_511 N N Anaplasma phagocytophilum 166 NC_007797 clade_511 N N Ehrlichia chaffeensis 783 AAIF01000035 clade_511 N OP Neorickettsia risticii 1349 CP001431 clade_511 N N Neorickettsia sennetsu 1350 NC_007798 clade_511 N N Pseudoramibacter alactolyticus 1606 AB036759 clade_512 N N Veillonella montpellierensis 1977 AF473836 clade_513 N N Veillonella sp. oral clone 1988 AY923118 clade_513 N N ASCA08 Veillonella sp. oral clone 1989 AY923122 clade_513 N N ASCB03 Inquilinus limosus 1012 NR_029046 clade_514 N N Sphingomonas sp. oral clone 1746 AY349412 clade_514 N N FZ016 Anaerococcus lactolyticus 145 ABYO01000217 clade_515 N N Anaerococcus prevotii 147 CP001708 clade_515 N N Anaerococcus sp. gpac104 152 AM176528 clade_515 N N Anaerococcus sp. gpac126 153 AM176530 clade_515 N N Anaerococcus sp. gpac155 154 AM176536 clade_515 N N Anaerococcus sp. gpac199 155 AM176539 clade_515 N N Anaerococcus tetradius 157 ACGC01000107 clade_515 N N Bacteroides coagulans 271 AB547639 clade_515 N N Clostridiales bacterium 534 HM587324 clade_515 N N 9403326 Clostridiales bacterium ph2 539 JN837487 clade_515 N N Peptostreptococcus sp. 9succ1 1457 X90471 clade_515 N N Peptostreptococcus sp. oral 1459 AB175072 clade_515 N N clone AP24 Tissierella praeacuta 1924 NR_044860 clade_515 N N Helicobacter canadensis 994 ABQS01000108 clade_518 N N Peptostreptococcus 1455 AY326462 clade_520 N N anaerobius Peptostreptococcus stomatis 1461 ADGQ01000048 clade_520 N N Bilophila wadsworthia 367 ADCP01000166 clade_521 N N Desulfovibrio vulgaris 761 NR_074897 clade_521 N N Actinomyces nasicola 64 AJ508455 clade_523 N N Cellulosimicrobium funkei 500 AY501364 clade_523 N N Lactococcus raffinolactis 1146 NR_044359 clade_524 N N Bacteroidales genomosp. P1 258 AY341819 clade_529 N N Bacteroidales genomosp. P2 259 DQ003613 clade_529 N N oral clone MB1_G13 Bacteroidales genomosp. P3 260 DQ003615 clade_529 N N oral clone MB1_G34 Bacteroidales genomosp. P4 261 DQ003617 clade_529 N N oral clone MB2_G17 Bacteroidales genomosp. P5 262 DQ003619 clade_529 N N oral clone MB2_P04 Bacteroidales genomosp. P6 263 DQ003634 clade_529 N N oral clone MB3_C19 Bacteroidales genomosp. P8 265 DQ003626 clade_529 N N oral clone MB4_G15 Bacteroidetes bacterium oral 333 HM099638 clade_530 N N taxon D27 Bacteroidetes bacterium oral 334 HM099643 clade_530 N N taxon F31 Bacteroidetes bacterium oral 335 HM099649 clade_530 N N taxon F44 Flavobacterium sp. NF2_1 885 FJ195988 clade_530 N N Myroides odoratimimus 1326 NR_042354 clade_530 N N Myroides sp. MY15 1327 GU253339 clade_530 N N Chlamydiales bacterium NS16 507 JN606076 clade_531 N N Chlamydophila pecorum 508 D88317 clade_531 N OP Parachlamydia sp. UWE25 1423 BX908798 clade_531 N N Fusobacterium russii 903 NR_044687 clade_532 N N Streptobacillus moniliformis 1784 NR_027615 clade_532 N N Eubacteriaceae bacterium 833 AY207060 clade_533 N N P4P_50 P4 Abiotrophia defectiva 1 ACIN02000016 clade_534 N N Abiotrophia sp. oral clone 3 AY207063 clade_534 N N P4PA_155 P1 Catonella genomosp. P1 oral 496 DQ003629 clade_534 N N clone MB5_P12 Catonella morbi 497 ACIL02000016 clade_534 N N Catonella sp. oral clone FL037 498 AY349369 clade_534 N N Eremococcus coleocola 818 AENN01000008 clade_534 N N Facklamia hominis 879 Y10772 clade_534 N N Granulicatella sp. M658_99_3 962 AJ271861 clade_534 N N Campylobacter coli 459 AAFL01000004 clade_535 N OP Campylobacter concisus 460 CP000792 clade_535 N OP Campylobacter fetus 462 ACLG01001177 clade_535 N OP Campylobacter jejuni 465 AL139074 clade_535 N Category-B Campylobacter upsaliensis 473 AEPU01000040 clade_535 N OP Atopobium minutum 183 HM007583 clade_539 N N Atopobium parvulum 184 CP001721 clade_539 N N Atopobium rimae 185 ACFE01000007 clade_539 N N Atopobium sp. BS2 186 HQ616367 clade_539 N N Atopobium sp. F0209 187 EU592966 clade_539 N N Atopobium sp. ICM42b10 188 HQ616393 clade_539 N N Atopobium sp. ICM57 189 HQ616400 clade_539 N N Atopobium vaginae 190 AEDQ01000024 clade_539 N N Coriobacteriaceae bacterium 677 JN809768 clade_539 N N BV3Ac1 Actinomyces naeslundii 63 X81062 clade_54 N N Actinomyces oricola 67 NR_025559 clade_54 N N Actinomyces oris 69 BABV01000070 clade_54 N N Actinomyces sp. 7400942 70 EU484334 clade_54 N N Actinomyces sp. ChDC B197 72 AF543275 clade_54 N N Actinomyces sp. GEJ15 73 GU561313 clade_54 N N Actinomyces sp. M2231_94_1 79 AJ234063 clade_54 N N Actinomyces sp. oral clone 83 AY349362 clade_54 N N GU067 Actinomyces sp. oral clone 85 AY349364 clade_54 N N IO077 Actinomyces sp. oral clone 86 AY349365 clade_54 N N IP073 Actinomyces sp. oral clone 88 AY349367 clade_54 N N JA063 Actinomyces sp. oral taxon 89 AFBL01000010 clade_54 N N 170 Actinomyces sp. oral taxon 90 AECW01000034 clade_54 N N 171 Actinomyces urogenitalis 95 ACFH01000038 clade_54 N N Actinomyces viscosus 96 ACRE01000096 clade_54 N N Orientia tsutsugamushi 1383 AP008981 clade_541 N OP Megamonas funiformis 1198 AB300988 clade_542 N N Megamonas hypermegale 1199 AJ420107 clade_542 N N Aeromicrobium marinum 102 NR_025681 clade_544 N N Aeromicrobium sp. JC14 103 JF824798 clade_544 N N Luteococcus sanguinis 1190 NR_025507 clade_544 N N Propionibacteriaceae 1568 EF599122 clade_544 N N bacterium NML 02_0265 Rhodococcus 1622 X80615 clade_546 N N corynebacterioides Rhodococcus erythropolis 1624 ACNO01000030 clade_546 N N Rhodococcus fascians 1625 NR_037021 clade_546 N N Segniliparus rotundus 1690 CP001958 clade_546 N N Segniliparus rugosus 1691 ACZI01000025 clade_546 N N Exiguobacterium acetylicum 878 FJ970034 clade_547 N N Macrococcus caseolyticus 1194 NR_074941 clade_547 N N Streptomyces sp. 1 AIP_2009 1890 FJ176782 clade_548 N N Streptomyces sp. SD 524 1892 EU544234 clade_548 N N Streptomyces sp. SD 528 1893 EU544233 clade_548 N N Streptomyces thermoviolaceus 1895 NR_027616 clade_548 N N Borrelia afzelii 388 ABCU01000001 clade_549 N OP Borrelia crocidurae 390 DQ057990 clade_549 N OP Borrelia duttonii 391 NC_011229 clade_549 N OP Borrelia hermsii 393 AY597657 clade_549 N OP Borrelia hispanica 394 DQ057988 clade_549 N OP Borrelia persica 395 HM161645 clade_549 N OP Borrelia recurrentis 396 AF107367 clade_549 N OP Borrelia spielmanii 398 ABKB01000002 clade_549 N OP Borrelia turicatae 399 NC_008710 clade_549 N OP Borrelia valaisiana 400 ABCY01000002 clade_549 N OP Providencia alcalifaciens 1586 ABXW01000071 clade_55 N N Providencia rettgeri 1587 AM040492 clade_55 N N Providencia rustigianii 1588 AM040489 clade_55 N N Providencia stuartii 1589 AF008581 clade_55 N N Treponema pallidum 1932 CP001752 clade_550 N OP Treponema phagedenis 1934 AEFH01000172 clade_550 N N Treponema sp. clone DDKL_4 1939 Y08894 clade_550 N N Acholeplasma laidlawii 17 NR_074448 clade_551 N N Mycoplasma putrefaciens 1323 U26055 clade_551 N N Mycoplasmataceae genomosp. 1325 DQ003614 clade_551 N N P1 oral clone MB1_G23 Spiroplasma insolitum 1750 NR_025705 clade_551 N N Collinsella intestinalis 660 ABXH02000037 clade_553 N N Collinsella stercoris 661 ABXJ01000150 clade_553 N N Collinsella tanakaei 662 AB490807 clade_553 N N Caminicella sporogenes 458 NR_025485 clade_554 N N Acidaminococcus fermentans 21 CP001859 clade_556 N N Acidaminococcus intestini 22 CP003058 clade_556 N N Acidaminococcus sp. D21 23 ACGB01000071 clade_556 N N Phascolarctobacterium 1462 NR_026111 clade_556 N N faecium Phascolarctobacterium sp. YIT 1463 AB490812 clade_556 N N 12068 Phascolarctobacterium 1464 AB490811 clade_556 N N succinatutens Acidithiobacillus ferrivorans 25 NR_074660 clade_557 N N Xanthomonadaceae bacterium 2015 EU313791 clade_557 N N NML 03_0222 Catabacter hongkongensis 494 AB671763 clade_558 N N Christensenella minuta 512 AB490809 clade_558 N N Clostridiales bacterium oral 536 AY207065 clade_558 N N clone P4PA_66 P1 Clostridiales bacterium oral 537 GQ422712 clade_558 N N taxon 093 Heliobacterium modesticaldum 1000 NR_074517 clade_560 N N Alistipes indistinctus 130 AB490804 clade_561 N N Bacteroidales bacterium ph8 257 JN837494 clade_561 N N Candidatus Sulcia muelleri 475 CP002163 clade_561 N N Cytophaga xylanolytica 742 FR733683 clade_561 N N Flavobacteriaceae genomosp. 884 AY278614 clade_561 N N C1 Gramella forsetii 958 NR_074707 clade_561 N N Sphingobacterium faecium 1740 NR_025537 clade_562 N N Sphingobacterium mizutaii 1741 JF708889 clade_562 N N Sphingobacterium multivorum 1742 NR_040953 clade_562 N N Sphingobacterium spiritivorum 1743 ACHA02000013 clade_562 N N Jonquetella anthropi 1017 ACOO02000004 clade_563 N N Pyramidobacter piscolens 1614 AY207056 clade_563 N N Synergistes genomosp. C1 1904 AY278615 clade_563 N N Synergistes sp. RMA 14551 1905 DQ412722 clade_563 N N Synergistetes bacterium 1906 GQ258968 clade_563 N N ADV897 Candidatus Arthromitus sp. 474 NR_074460 clade_564 N N SFB_mouse_Yit Gracilibacter thermotolerans 957 NR_043559 clade_564 N N Brachyspira aalborgi 404 FM178386 clade_565 N N Brachyspira sp. HIS3 406 FM178387 clade_565 N N Brachyspira sp. HIS4 407 FM178388 clade_565 N N Brachyspira sp. HIS5 408 FM178389 clade_565 N N Adlercreutzia equolifaciens 97 AB306661 clade_566 N N Coriobacteriaceae bacterium 678 CAEM01000062 clade_566 N N JC110 Coriobacteriaceae bacterium 679 JN837493 clade_566 N N phI Cryptobacterium curtum 740 GQ422741 clade_566 N N Eggerthella sinensis 779 AY321958 clade_566 N N Eggerthella sp. 1_3_56FAA 780 ACWN01000099 clade_566 N N Eggerthella sp. HGA1 781 AEXR01000021 clade_566 N N Eggerthella sp. YY7918 782 AP012211 clade_566 N N Gordonibacter pamelaeae 680 AM886059 clade_566 N N Gordonibacter pamelaeae 956 FP929047 clade_566 N N Slackia equolifaciens 1732 EU377663 clade_566 N N Slackia exigua 1733 ACUX01000029 clade_566 N N Slackia faecicanis 1734 NR_042220 clade_566 N N Slackia heliotrinireducens 1735 NR_074439 clade_566 N N Slackia isoflavoniconvertens 1736 AB566418 clade_566 N N Slackia piriformis 1737 AB490806 clade_566 N N Slackia sp. NATTS 1738 AB505075 clade_566 N N Chlamydiales bacterium NS13 506 JN606075 clade_567 N N Victivallaceae bacterium NML 2003 FJ394915 clade_567 N N 080035 Victivallis vadensis 2004 ABDE02000010 clade_567 N N Streptomyces griseus 1889 NR_074787 clade_573 N N Streptomyces sp. SD 511 1891 EU544231 clade_573 N N Streptomyces sp. SD 534 1894 EU544232 clade_573 N N Cloacibacillus evryensis 530 GQ258966 clade_575 N N Deferribacteres sp. oral clone 743 AY349370 clade_575 N N JV001 Deferribacteres sp. oral clone 745 AY349372 clade_575 N N JV023 Synergistetes bacterium 1907 GQ258969 clade_575 N N LBVCM1157 Synergistetes bacterium oral 1909 GU410752 clade_575 N N taxon 362 Synergistetes bacterium oral 1910 GU430992 clade_575 N N taxon D48 Peptococcus sp. oral clone 1439 AY349389 clade_576 N N JM048 Helicobacter winghamensis 999 ACDO01000013 clade_577 N N Wolinella succinogenes 2014 BX571657 clade_577 N N Olsenella genomosp. C1 1368 AY278623 clade_578 N N Olsenella profusa 1369 FN178466 clade_578 N N Olsenella sp. F0004 1370 EU592964 clade_578 N N Olsenella sp. oral taxon 809 1371 ACVE01000002 clade_578 N N Olsenella uli 1372 CP002106 clade_578 N N Nocardiopsis dassonvillei 1356 CP002041 clade_579 N N Peptococcus niger 1438 NR_029221 clade_580 N N Peptococcus sp. oral taxon 1440 GQ422727 clade_580 N N 167 Akkermansia muciniphila 118 CP001071 clade_583 N N Opitutus terrae 1373 NR_074978 clade_583 N N Clostridiales bacterium oral 538 HM099644 clade_584 N N taxon F32 Leptospira borgpetersenii 1161 NC_008508 clade_585 N OP Leptospira broomii 1162 NR_043200 clade_585 N OP Leptospira interrogans 1163 NC_005823 clade_585 N OP Methanobrevibacter 1213 NR_044789 clade_587 N N gottschalkii Methanobrevibacter millerae 1214 NR_042785 clade_587 N N Methanobrevibacter oralis 1216 HE654003 clade_587 N N Methanobrevibacter thaueri 1219 NR_044787 clade_587 N N Methanobrevibacter smithii 1218 ABYV02000002 clade_588 N N Deinococcus radiodurans 746 AE000513 clade_589 N N Deinococcus sp. R_43890 747 FR682752 clade_589 N N Thermus aquaticus 1923 NR_025900 clade_589 N N Actinomyces sp. c109 81 AB167239 clade_590 N N Syntrophomonadaceae 1912 AY341821 clade_590 N N genomosp. P1 Anaerobaculum 141 ACJX02000009 clade_591 N N hydrogeniformans Microcystis aeruginosa 1246 NC_010296 clade_592 N N Prochlorococcus marinus 1567 CP000551 clade_592 N N Methanobrevibacter 1208 NR_028779 clade_593 N N acididurans Methanobrevibacter 1209 NR_042783 clade_593 N N arboriphilus Methanobrevibacter curvatus 1210 NR_044796 clade_593 N N Methanobrevibacter cuticularis 1211 NR_044776 clade_593 N N Methanobrevibacter filiformis 1212 NR_044801 clade_593 N N Methanobrevibacter woesei 1220 NR_044788 clade_593 N N Roseiflexus castenholzii 1642 CP000804 clade_594 N N Methanobrevibacter olleyae 1215 NR_043024 clade_595 N N Methanobrevibacter 1217 NR_042784 clade_595 N N ruminantium Methanobrevibacter wolinii 1221 NR_044790 clade_595 N N Methanosphaera stadtmanae 1222 AY196684 clade_595 N N Chloroflexi genomosp. P1 511 AY331414 clade_596 N N Halorubrum lipolyticum 992 AB477978 clade_597 N N Methanobacterium formicicum 1207 NR_025028 clade_597 N N Acidilobus saccharovorans 24 AY350586 clade_598 N N Hyperthermus butylicus 1006 CP000493 clade_598 N N Ignicoccus islandicus 1011 X99562 clade_598 N N Metallosphaera sedula 1206 D26491 clade_598 N N Thermofilum pendens 1922 X14835 clade_598 N N Prevotella melaninogenica 1506 CP002122 clade_6 N N Prevotella sp. ICM1 1520 HQ616385 clade_6 N N Prevotella sp. oral clone 1535 AY349393 clade_6 N N FU048 Prevotella sp. oral clone GI030 1537 AY349395 clade_6 N N Prevotella sp. SEQ116 1526 JN867246 clade_6 N N Streptococcus anginosus 1787 AECT01000011 clade_60 N N Streptococcus milleri 1812 X81023 clade_60 N N Streptococcus sp. 16362 1829 JN590019 clade_60 N N Streptococcus sp. 69130 1832 X78825 clade_60 N N Streptococcus sp. AC15 1833 HQ616356 clade_60 N N Streptococcus sp. CM7 1839 HQ616373 clade_60 N N Streptococcus sp. OBRC6 1847 HQ616352 clade_60 N N Burkholderia ambifaria 442 AAUZ01000009 clade_61 N OP Burkholderia cenocepacia 443 AAHI01000060 clade_61 N OP Burkholderia cepacia 444 NR_041719 clade_61 N OP Burkholderia mallei 445 CP000547 clade_61 N Category-B Burkholderia multivorans 446 NC_010086 clade_61 N OP Burkholderia oklahomensis 447 DQ108388 clade_61 N OP Burkholderia pseudomallei 448 CP001408 clade_61 N Category-B Burkholderia rhizoxinica 449 HQ005410 clade_61 N OP Burkholderia sp. 383 450 CP000151 clade_61 N OP Burkholderia xenovorans 451 U86373 clade_61 N OP Prevotella buccae 1488 ACRB01000001 clade_62 N N Prevotella genomosp. P8 oral 1498 DQ003622 clade_62 N N clone MB3_P13 Prevotella sp. oral clone 1536 AY349394 clade_62 N N FW035 Prevotella bivia 1486 ADFO01000096 clade_63 N N Prevotella disiens 1494 AEDO01000026 clade_64 N N Bacteroides faecis 276 GQ496624 clade_65 N N Bacteroides fragilis 279 AP006841 clade_65 N N Bacteroides nordii 285 NR_043017 clade_65 N N Bacteroides salyersiae 292 EU136690 clade_65 N N Bacteroides sp. 1_1_14 293 ACRP01000155 clade_65 N N Bacteroides sp. 1_1_6 295 ACIC01000215 clade_65 N N Bacteroides sp. 2_1_56FAA 298 ACWI01000065 clade_65 N N Bacteroides sp. AR29 316 AF139525 clade_65 N N Bacteroides sp. B2 317 EU722733 clade_65 N N Bacteroides thetaiotaomicron 328 NR_074277 clade_65 N N Actinobacillus minor 45 ACFT01000025 clade_69 N N Haemophilus parasuis 978 GU226366 clade_69 N N Vibrio cholerae 1996 AAUR01000095 clade_71 N Category-B Vibrio fluvialis 1997 X76335 clade_71 N Category-B Vibrio furnissii 1998 CP002377 clade_71 N Category-B Vibrio mimicus 1999 ADAF01000001 clade_71 N Category-B Vibrio parahaemolyticus 2000 AAWQ01000116 clade_71 N Category-B Vibrio sp. RC341 2001 ACZT01000024 clade_71 N Category-B Vibrio vulnificus 2002 AE016796 clade_71 N Category-B Lactobacillus acidophilus 1067 CP000033 clade_72 N N Lactobacillus amylolyticus 1069 ADNY01000006 clade_72 N N Lactobacillus amylovorus 1070 CP002338 clade_72 N N Lactobacillus crispatus 1078 ACOG01000151 clade_72 N N Lactobacillus delbrueckii 1080 CP002341 clade_72 N N Lactobacillus helveticus 1088 ACLM01000202 clade_72 N N Lactobacillus kalixensis 1094 NR_029083 clade_72 N N Lactobacillus kefiranofaciens 1095 NR_042440 clade_72 N N Lactobacillus leichmannii 1098 JX986966 clade_72 N N Lactobacillus sp. 66c 1120 FR681900 clade_72 N N Lactobacillus sp. KLDS 1.0701 1122 EU600905 clade_72 N N Lactobacillus sp. KLDS 1.0712 1130 EU600916 clade_72 N N Lactobacillus sp. oral clone 1136 AY349383 clade_72 N N HT070 Lactobacillus ultunensis 1139 ACGU01000081 clade_72 N N Prevotella intermedia 1502 AF414829 clade_81 N N Prevotella nigrescens 1511 AFPX01000069 clade_81 N N Prevotella pallens 1515 AFPY01000135 clade_81 N N Prevotella sp. oral taxon 310 1551 GQ422737 clade_81 N N Prevotella genomosp. C1 1495 AY278624 clade_82 N N Prevotella sp. CM38 1519 HQ610181 clade_82 N N Prevotella sp. oral taxon 317 1552 ACQH01000158 clade_82 N N Prevotella sp. SG12 1527 GU561343 clade_82 N N Prevotella denticola 1493 CP002589 clade_83 N N Prevotella genomosp. P7 oral 1497 DQ003620 clade_83 N N clone MB2_P31 Prevotella histicola 1501 JN867315 clade_83 N N Prevotella multiformis 1508 AEWX01000054 clade_83 N N Prevotella sp. JCM 6330 1522 AB547699 clade_83 N N Prevotella sp. oral clone GI059 1539 AY349397 clade_83 N N Prevotella sp. oral taxon 782 1555 GQ422745 clade_83 N N Prevotella sp. oral taxon G71 1559 GU432180 clade_83 N N Prevotella sp. SEQ065 1524 JN867234 clade_83 N N Prevotella veroralis 1565 ACVA01000027 clade_83 N N Bacteroides acidifaciens 266 NR_028607 clade_85 N N Bacteroides cellulosilyticus 269 ACCH01000108 clade_85 N N Bacteroides clarus 270 AFBM01000011 clade_85 N N Bacteroides eggerthii 275 ACWG01000065 clade_85 N N Bacteroides oleiciplenus 286 AB547644 clade_85 N N Bacteroides pyogenes 290 NR_041280 clade_85 N N Bacteroides sp. 315_5 300 FJ848547 clade_85 N N Bacteroides sp. 31SF15 301 AJ583248 clade_85 N N Bacteroides sp. 31SF18 302 AJ583249 clade_85 N N Bacteroides sp. 35AE31 303 AJ583244 clade_85 N N Bacteroides sp. 35AE37 304 AJ583245 clade_85 N N Bacteroides sp. 35BE34 305 AJ583246 clade_85 N N Bacteroides sp. 35BE35 306 AJ583247 clade_85 N N Bacteroides sp. WH2 324 AY895180 clade_85 N N Bacteroides sp. XB12B 325 AM230648 clade_85 N N Bacteroides stercoris 327 ABFZ02000022 clade_85 N N Actinobacillus 46 NR_074857 clade_88 N N pleuropneumoniae Actinobacillus ureae 48 AEVG01000167 clade_88 N N Haemophilus aegyptius 969 AFBC01000053 clade_88 N N Haemophilus ducreyi 970 AE017143 clade_88 N OP Haemophilus haemolyticus 973 JN175335 clade_88 N N Haemophilus influenzae 974 AADP01000001 clade_88 N OP Haemophilus 975 GU561425 clade_88 N N parahaemolyticus Haemophilus parainfluenzae 976 AEWU01000024 clade_88 N N Haemophilus 977 M75076 clade_88 N N paraphrophaemolyticus Haemophilus somnus 979 NC_008309 clade_88 N N Haemophilus sp. 70334 980 HQ680854 clade_88 N N Haemophilus sp. HK445 981 FJ685624 clade_88 N N Haemophilus sp. oral clone 982 AY923117 clade_88 N N ASCA07 Haemophilus sp. oral clone 983 AY923147 clade_88 N N ASCG06 Haemophilus sp. oral clone 984 AY005034 clade_88 N N BJ021 Haemophilus sp. oral clone 985 AY005033 clade_88 N N BJ095 Haemophilus sp. oral taxon 987 AGRK01000004 clade_88 N N 851 Haemophilus sputorum 988 AFNK01000005 clade_88 N N Histophilus somni 1003 AF549387 clade_88 N N Mannheimia haemolytica 1195 ACZX01000102 clade_88 N N Pasteurella bettyae 1433 L06088 clade_88 N N Moellerella wisconsensis 1253 JN175344 clade_89 N N Morganella morganii 1265 AJ301681 clade_89 N N Morganella sp. JB_T16 1266 AJ781005 clade_89 N N Proteus mirabilis 1582 ACLE01000013 clade_89 N N Proteus penneri 1583 ABVP01000020 clade_89 N N Proteus sp. HS7514 1584 DQ512963 clade_89 N N Proteus vulgaris 1585 AJ233425 clade_89 N N Oribacterium sinus 1374 ACKX01000142 clade_90 N N Oribacterium sp. ACB1 1375 HM120210 clade_90 N N Oribacterium sp. ACB7 1376 HM120211 clade_90 N N Oribacterium sp. CM12 1377 HQ616374 clade_90 N N Oribacterium sp. ICM51 1378 HQ616397 clade_90 N N Oribacterium sp. OBRC12 1379 HQ616355 clade_90 N N Oribacterium sp. oral taxon 1382 AFIH01000001 clade_90 N N 108 Actinobacillus 44 AY362885 clade_92 N N actinomycetemcomitans Actinobacillus succinogenes 47 CP000746 clade_92 N N Aggregatibacter 112 CP001733 clade_92 N N actinomycetemcomitans Aggregatibacter aphrophilus 113 CP001607 clade_92 N N Aggregatibacter segnis 114 AEPS01000017 clade_92 N N Averyella dalhousiensis 194 DQ481464 clade_92 N N Bisgaard Taxon 368 AY683487 clade_92 N N Bisgaard Taxon 369 AY683489 clade_92 N N Bisgaard Taxon 370 AY683491 clade_92 N N Bisgaard Taxon 371 AY683492 clade_92 N N Buchnera aphidicola 440 NR_074609 clade_92 N N Cedecea davisae 499 AF493976 clade_92 N N Citrobacter amalonaticus 517 FR870441 clade_92 N N Citrobacter braakii 518 NR_028687 clade_92 N N Citrobacter farmeri 519 AF025371 clade_92 N N Citrobacter freundii 520 NR_028894 clade_92 N N Citrobacter gillenii 521 AF025367 clade_92 N N Citrobacter koseri 522 NC_009792 clade_92 N N Citrobacter murliniae 523 AF025369 clade_92 N N Citrobacter rodentium 524 NR_074903 clade_92 N N Citrobacter sedlakii 525 AF025364 clade_92 N N Citrobacter sp. 30_2 526 ACDJ01000053 clade_92 N N Citrobacter sp. KMSI_3 527 GQ468398 clade_92 N N Citrobacter werkmanii 528 AF025373 clade_92 N N Citrobacter youngae 529 ABWL02000011 clade_92 N N Cronobacter malonaticus 737 GU122174 clade_92 N N Cronobacter sakazakii 738 NC_009778 clade_92 N N Cronobacter turicensis 739 FN543093 clade_92 N N Enterobacter aerogenes 786 AJ251468 clade_92 N N Enterobacter asburiae 787 NR_024640 clade_92 N N Enterobacter cancerogenus 788 Z96078 clade_92 N N Enterobacter cloacae 789 FP929040 clade_92 N N Enterobacter cowanii 790 NR_025566 clade_92 N N Enterobacter hormaechei 791 AFHR01000079 clade_92 N N Enterobacter sp. 247BMC 792 HQ122932 clade_92 N N Enterobacter sp. 638 793 NR_074777 clade_92 N N Enterobacter sp. JC163 794 JN657217 clade_92 N N Enterobacter sp. SCSS 795 HM007811 clade_92 N N Enterobacter sp. TSE38 796 HM156134 clade_92 N N Enterobacteriaceae bacterium 797 ADCU01000033 clade_92 N N 9_2_54FAA Enterobacteriaceae bacterium 798 AJ489826 clade_92 N N CF01Ent_1 Enterobacteriaceae bacterium 799 AY538694 clade_92 N N Smarlab 3302238 Escherichia albertii 824 ABKX01000012 clade_92 N N Escherichia coli 825 NC_008563 clade_92 N Category-B Escherichia fergusonii 826 CU928158 clade_92 N N Escherichia hermannii 827 HQ407266 clade_92 N N Escherichia sp. 1_1_43 828 ACID01000033 clade_92 N N Escherichia sp. 4_1_40B 829 ACDM02000056 clade_92 N N Escherichia sp. B4 830 EU722735 clade_92 N N Escherichia vulneris 831 NR_041927 clade_92 N N Ewingella americana 877 JN175329 clade_92 N N Haemophilus genomosp. P2 971 DQ003621 clade_92 N N oral clone MB3_C24 Haemophilus genomosp. P3 972 DQ003635 clade_92 N N oral clone MB3_C38 Haemophilus sp. oral clone 986 AY349380 clade_92 N N JM053 Hafnia alvei 989 DQ412565 clade_92 N N Klebsiella oxytoca 1024 AY292871 clade_92 N OP Klebsiella pneumoniae 1025 CP000647 clade_92 N OP Klebsiella sp. AS10 1026 HQ616362 clade_92 N N Klebsiella sp. Co9935 1027 DQ068764 clade_92 N N Klebsiella sp. enrichment 1036 HM195210 clade_92 N N culture clone SRC_DSD25 Klebsiella sp. OBRC7 1028 HQ616353 clade_92 N N Klebsiella sp. SP_BA 1029 FJ999767 clade_92 N N Klebsiella sp. SRC_DSD1 1033 GU797254 clade_92 N N Klebsiella sp. SRC_DSD11 1030 GU797263 clade_92 N N Klebsiella sp. SRC_DSD12 1031 GU797264 clade_92 N N Klebsiella sp. SRC_DSD15 1032 GU797267 clade_92 N N Klebsiella sp. SRC_DSD2 1034 GU797253 clade_92 N N Klebsiella sp. SRC_DSD6 1035 GU797258 clade_92 N N Klebsiella variicola 1037 CP001891 clade_92 N N Kluyvera ascorbata 1038 NR_028677 clade_92 N N Kluyvera cryocrescens 1039 NR_028803 clade_92 N N Leminorella grimontii 1159 AJ233421 clade_92 N N Leminorella richardii 1160 HF558368 clade_92 N N Pantoea agglomerans 1409 AY335552 clade_92 N N Pantoea ananatis 1410 CP001875 clade_92 N N Pantoea brenneri 1411 EU216735 clade_92 N N Pantoea citrea 1412 EF688008 clade_92 N N Pantoea conspicua 1413 EU216737 clade_92 N N Pantoea septica 1414 EU216734 clade_92 N N Pasteurella dagmatis 1434 ACZR01000003 clade_92 N N Pasteurella multocida 1435 NC_002663 clade_92 N N Plesiomonas shigelloides 1469 X60418 clade_92 N N Raoultella ornithinolytica 1617 AB364958 clade_92 N N Raoultella planticola 1618 AF129443 clade_92 N N Raoultella terrigena 1619 NR_037085 clade_92 N N Salmonella bongori 1683 NR_041699 clade_92 N Category-B Salmonella enterica 1672 NC_011149 clade_92 N Category-B Salmonella enterica 1673 NC_011205 clade_92 N Category-B Salmonella enterica 1674 DQ344532 clade_92 N Category-B Salmonella enterica 1675 ABEH02000004 clade_92 N Category-B Salmonella enterica 1676 ABAK02000001 clade_92 N Category-B Salmonella enterica 1677 NC_011080 clade_92 N Category-B Salmonella enterica 1678 EU118094 clade_92 N Category-B Salmonella enterica 1679 NC_011094 clade_92 N Category-B Salmonella enterica 1680 AE014613 clade_92 N Category-B Salmonella enterica 1682 ABFH02000001 clade_92 N Category-B Salmonella enterica 1684 ABEM01000001 clade_92 N Category-B Salmonella enterica 1685 ABAM02000001 clade_92 N Category-B Salmonella typhimurium 1681 DQ344533 clade_92 N Category-B Salmonella typhimurium 1686 AF170176 clade_92 N Category-B Serratia fonticola 1718 NR_025339 clade_92 N N Serratia liquefaciens 1719 NR_042062 clade_92 N N Serratia marcescens 1720 GU826157 clade_92 N N Serratia odorifera 1721 ADBY01000001 clade_92 N N Serratia proteamaculans 1722 AAUN01000015 clade_92 N N Shigella boydii 1724 AAKA01000007 clade_92 N Category-B Shigella dysenteriae 1725 NC_007606 clade_92 N Category-B Shigella flexneri 1726 AE005674 clade_92 N Category-B Shigella sonnei 1727 NC_007384 clade_92 N Category-B Tatumella ptyseos 1916 NR_025342 clade_92 N N Trabulsiella guamensis 1925 AY373830 clade_92 N N Yersinia aldovae 2019 AJ871363 clade_92 N OP Yersinia aleksiciae 2020 AJ627597 clade_92 N OP Yersinia bercovieri 2021 AF366377 clade_92 N OP Yersinia enterocolitica 2022 FR729477 clade_92 N Category-B Yersinia frederiksenii 2023 AF366379 clade_92 N OP Yersinia intermedia 2024 AF366380 clade_92 N OP Yersinia kristensenii 2025 ACCA01000078 clade_92 N OP Yersinia mollaretii 2026 NR_027546 clade_92 N OP Yersinia pestis 2027 AE013632 clade_92 N Category-A Yersinia pseudotuberculosis 2028 NC_009708 clade_92 N OP Yersinia rohdei 2029 ACCD01000071 clade_92 N OP Yokenella regensburgei 2030 AB273739 clade_92 N N Conchiformibius kuhniae 669 NR_041821 clade_94 N N Morococcus cerebrosus 1267 JN175352 clade_94 N N Neisseria bacilliformis 1328 AFAY01000058 clade_94 N N Neisseria cinerea 1329 ACDY01000037 clade_94 N N Neisseria flavescens 1331 ACQV01000025 clade_94 N N Neisseria gonorrhoeae 1333 CP002440 clade_94 N OP Neisseria lactamica 1334 ACEQ01000095 clade_94 N N Neisseria macacae 1335 AFQE01000146 clade_94 N N Neisseria meningitidis 1336 NC_003112 clade_94 N OP Neisseria mucosa 1337 ACDX01000110 clade_94 N N Neisseria pharyngis 1338 AJ239281 clade_94 N N Neisseria polysaccharea 1339 ADBE01000137 clade_94 N N Neisseria sicca 1340 ACK002000016 clade_94 N N Neisseria sp. KEM232 1341 GQ203291 clade_94 N N Neisseria sp. oral clone AP132 1344 AY005027 clade_94 N N Neisseria sp. oral strain B33KA 1346 AY005028 clade_94 N N Neisseria sp. oral taxon 014 1347 ADEA01000039 clade_94 N N Neisseria sp. TM10_1 1343 DQ279352 clade_94 N N Neisseria subflava 1348 ACEO01000067 clade_94 N N Okadaella gastrococcus 1365 HQ699465 clade_98 N N Streptococcus agalactiae 1785 AAJO01000130 clade_98 N N Streptococcus alactolyticus 1786 NR_041781 clade_98 N N Streptococcus australis 1788 AEQR01000024 clade_98 N N Streptococcus bovis 1789 AEEL01000030 clade_98 N N Streptococcus canis 1790 AJ413203 clade_98 N N Streptococcus constellatus 1791 AY277942 clade_98 N N Streptococcus cristatus 1792 AEVC01000028 clade_98 N N Streptococcus dysgalactiae 1794 AP010935 clade_98 N N Streptococcus equi 1795 CP001129 clade_98 N N Streptococcus equinus 1796 AEVB01000043 clade_98 N N Streptococcus gallolyticus 1797 FR824043 clade_98 N N Streptococcus genomosp. C1 1798 AY278629 clade_98 N N Streptococcus genomosp. C2 1799 AY278630 clade_98 N N Streptococcus genomosp. C3 1800 AY278631 clade_98 N N Streptococcus genomosp. C4 1801 AY278632 clade_98 N N Streptococcus genomosp. C5 1802 AY278633 clade_98 N N Streptococcus genomosp. C6 1803 AY278634 clade_98 N N Streptococcus genomosp. C7 1804 AY278635 clade_98 N N Streptococcus genomosp. C8 1805 AY278609 clade_98 N N Streptococcus gordonii 1806 NC_009785 clade_98 N N Streptococcus infantarius 1807 ABJK02000017 clade_98 N N Streptococcus infantis 1808 AFNN01000024 clade_98 N N Streptococcus intermedius 1809 NR_028736 clade_98 N N Streptococcus lutetiensis 1810 NR_037096 clade_98 N N Streptococcus massiliensis 1811 AY769997 clade_98 N N Streptococcus mitis 1813 AM157420 clade_98 N N Streptococcus oligofermentans 1815 AY099095 clade_98 N N Streptococcus oralis 1816 ADMV01000001 clade_98 N N Streptococcus parasanguinis 1817 AEKM01000012 clade_98 N N Streptococcus pasteurianus 1818 AP012054 clade_98 N N Streptococcus peroris 1819 AEVF01000016 clade_98 N N Streptococcus pneumoniae 1820 AE008537 clade_98 N N Streptococcus porcinus 1821 EF121439 clade_98 N N Streptococcus 1822 FJ827123 clade_98 N N pseudopneumoniae Streptococcus pseudoporcinus 1823 AENS01000003 clade_98 N N Streptococcus pyogenes 1824 AE006496 clade_98 N OP Streptococcus ratti 1825 X58304 clade_98 N N Streptococcus sanguinis 1827 NR_074974 clade_98 N N Streptococcus sinensis 1828 AF432857 clade_98 N N Streptococcus sp. 2_1_36FAA 1831 ACOI01000028 clade_98 N N Streptococcus sp. 2285_97 1830 AJ131965 clade_98 N N Streptococcus sp. ACS2 1834 HQ616360 clade_98 N N Streptococcus sp. AS20 1835 HQ616366 clade_98 N N Streptococcus sp. BS35a 1836 HQ616369 clade_98 N N Streptococcus sp. C150 1837 ACRI01000045 clade_98 N N Streptococcus sp. CM6 1838 HQ616372 clade_98 N N Streptococcus sp. ICM10 1840 HQ616389 clade_98 N N Streptococcus sp. ICM12 1841 HQ616390 clade_98 N N Streptococcus sp. ICM2 1842 HQ616386 clade_98 N N Streptococcus sp. ICM4 1844 HQ616387 clade_98 N N Streptococcus sp. ICM45 1843 HQ616394 clade_98 N N Streptococcus sp. M143 1845 ACRK01000025 clade_98 N N Streptococcus sp. M334 1846 ACRL01000052 clade_98 N N Streptococcus sp. oral clone 1849 AY923121 clade_98 N N ASB02 Streptococcus sp. oral clone 1850 DQ272504 clade_98 N N ASCA03 Streptococcus sp. oral clone 1851 AY923116 clade_98 N N ASCA04 Streptococcus sp. oral clone 1852 AY923119 clade_98 N N ASCA09 Streptococcus sp. oral clone 1853 AY923123 clade_98 N N ASCB04 Streptococcus sp. oral clone 1854 AY923124 clade_98 N N ASCB06 Streptococcus sp. oral clone 1855 AY923127 clade_98 N N ASCC04 Streptococcus sp. oral clone 1856 AY923128 clade_98 N N ASCC05 Streptococcus sp. oral clone 1857 DQ272507 clade_98 N N ASCC12 Streptococcus sp. oral clone 1858 AY923129 clade_98 N N ASCD01 Streptococcus sp. oral clone 1859 AY923130 clade_98 N N ASCD09 Streptococcus sp. oral clone 1860 DQ272509 clade_98 N N ASCD10 Streptococcus sp. oral clone 1861 AY923134 clade_98 N N ASCE03 Streptococcus sp. oral clone 1862 AY953253 clade_98 N N ASCE04 Streptococcus sp. oral clone 1863 DQ272510 clade_98 N N ASCE05 Streptococcus sp. oral clone 1864 AY923135 clade_98 N N ASCE06 Streptococcus sp. oral clone 1865 AY923136 clade_98 N N ASCE09 Streptococcus sp. oral clone 1866 AY923137 clade_98 N N ASCE10 Streptococcus sp. oral clone 1867 AY923138 clade_98 N N ASCE12 Streptococcus sp. oral clone 1868 AY923140 clade_98 N N ASCF05 Streptococcus sp. oral clone 1869 AY953255 clade_98 N N ASCF07 Streptococcus sp. oral clone 1870 AY923142 clade_98 N N ASCF09 Streptococcus sp. oral clone 1871 AY923145 clade_98 N N ASCG04 Streptococcus sp. oral clone 1872 AY005042 clade_98 N N BW009 Streptococcus sp. oral clone 1873 AY005044 clade_98 N N CH016 Streptococcus sp. oral clone 1874 AY349413 clade_98 N N GK051 Streptococcus sp. oral clone 1875 AY349414 clade_98 N N GM006 Streptococcus sp. oral clone 1876 AY207051 clade_98 N N P2PA_41 P2 Streptococcus sp. oral clone 1877 AY207064 clade_98 N N P4PA_30 P4 Streptococcus sp. oral taxon 1878 AEEP01000019 clade_98 N N 071 Streptococcus sp. oral taxon 1879 GU432132 clade_98 N N G59 Streptococcus sp. oral taxon 1880 GU432146 clade_98 N N G62 Streptococcus sp. oral taxon 1881 GU432150 clade_98 N N G63 Streptococcus suis 1882 FM252032 clade_98 N N Streptococcus thermophilus 1883 CP000419 clade_98 N N Streptococcus salivarius 1826 AGBV01000001 clade_98 N N Streptococcus uberis 1884 HQ391900 clade_98 N N Streptococcus urinalis 1885 DQ303194 clade_98 N N Streptococcus vestibularis 1886 AEKO01000008 clade_98 N N Streptococcus viridans 1887 AF076036 clade_98 N N Synergistetes bacterium oral 1908 GU227192 clade_98 N N clone 03 5 D05

TABLE X4 Spore-forming Bacterial Species Alkaliphilus metalliredigens Ammonifex degensii Anaerofustis stercorihominis Anaerostipes caccae Anaerotruncus colihominis Bacillus amyloliquefaciens Bacillus anthracis Bacillus cellulosilyticus Bacillus cereus Bacillus clausii Bacillus coagulans Bacillus cytotoxicus Bacillus halodurans Bacillus licheniformis Bacillus pumilus Bacillus subtilis Bacillus thuringiensis Bacillus weihenstephanensis Blautia hansenii Brevibacillus brevis Bryantella formatexigens Caldicellulosiruptor saccharolyticus Candidatus Desulforudis audaxviato Carboxydibrachium pacificum Carboxydothermus hydrogenoformans Clostridium acetobutylicum Clostridium asparagiforme Clostridium bartlettii Clostridium beijerinckii Clostridium bolteae Clostridium botulinum A str. ATCC 19397 Clostridium botulinum B str. Eklund 17B Clostridium butyricum pathogenic E4 str. BoNT BL5262 Clostridium Carboxidivorans Clostridium cellulolyticum Clostridium cellulovorans Clostridium difficile Clostridium hathewayi Clostridium hylemonae Clostridium kluyveri Clostridium leptum Clostridium methylpentosum Clostridium nexile Clostridium novyi NT Clostridium papyrosolvens Clostridium perfringens Clostridium phytofermentans ISDg Clostridium scindens Clostridium sp. 7_2_43FAA Clostridium sporogenes Clostridium tetani Clostridium thermocellum Coprococcus comes Desulfotomaculum reducens Dorea longicatena Eubacterium eligens Eubacterium hallii Eubacterium rectale Eubacterium ventriosum Faecalibacterium prausnitzii Geobacillus kaustophilus Geobacillus sp. G11MC16 Geobacillus thermodenitrificans Heliobacterium modesticaldum Lysinibacillus sphaericus Oceanobacillus iheyensis Paenibacillus sp. JDR-2 Pelotomaculum thermopropionicum Roseburia intestinalis Ruminococcus bromii Ruminococcus gnavus Ruminococcus obeum Ruminococcus torques Subdoligranulum variabile Symbiobacterium thermophilum Thermoanaerobacter italicus Thermoanaerobacter tengcongensis Thermoanaerobacterium thermosaccharolyticum Thermosinus carboxydivorans

TABLE 2 Species isolated from ethanol treated spore preparation preparation before (left) and after (right) CsCl gradient step ethanol treated, ethanol treated gradient purified Isolates spore preparation spore preparation Bacillus coagulans 7 2 Blautia luti 1 1 Blautia sp 14 13 Blautia wexlerae 3 1 Ruminococcus obeum 4 2 Clostridiales sp 1 2 Clostridium aerotolerans 1 2 Clostridium disporicum 0 1 Clostridium sp 1 1 Clostridium symbiosum 0 1 Dorea longicatena 8 6 Eubacterium cellulosolvens 1 0 Eubacterium ventriosum 2 2 Gemmiger formicilis 0 1 Robinsoniella peoriensis 0 1 Roseburia hominis 3 6 Roseburia intestinalis 9 7 Ruminococcus sp 5 2 Syntrophococcus sucromutans 1 1 Turicibacter sanguinis 3 4 Clostridiales sp 7 9 Clostridium bartlettii 8 11 Clostridium irregulare 0 1 Clostridium sordellii 4 6 Lachnospiraceae sp 1 0

TABLE 3 Mortality and weight change in mice challenged with C. difficile with or without ethanol treated, spore product treatment. mortality % weight change Test article (n = 10) on Day 3 vehicle (negative control) 20% −10.5%  Donor feces (positive control) 0 −0.1%  EtOH-treated feces 1x 0 2.3% EtOH-treated feces 0.1x 0 2.4% EtOH-treated feces 0.01x 0  −3% heat-treated feces 0 0.1%

TABLE 4 16S rDNA identified spore forming species from picked colony plates. Treatment Species No. isolates 70deg 1 h Clostridium_celatum 4 70deg 1 h Clostridium_clostridioform 1 70deg 1 h Clostridium_hylemonae 1 70deg 1 h Clostridium_paraputrificum 3 70deg 1 h Clostridium_sp_D5 1 70deg 1 h Clostridium_symbiosum 1 80deg 1 h Clostridium_bartlettii 6 80deg 1 h Clostridium_butyricum 1 80deg 1 h Clostridium_paraputrificum 5 80deg 1 h Coprobacillus_sp_D7 1 80deg 1 h Eubacterium_sp_WAL_14571 1 80deg 1 h Ruminococcus_bromii 1 90deg 1 h Clostridium_butyricum 1 90deg 10 min Ruminococcus_bromii 1 90deg 10 min Anaerotruncus_colihominis 2 90deg 10 min Clostridium_bartlettii 1 100deg 10 min Ruminococcus_bromii 1

TABLE 5 Spore-forming species identified in ethanol treated or heat treated samples and not identified in untreated samples isolated from isolated from isolated from Species untreated EtOH-treated heat-treated Acetivibrio ethanolgignens X Anaerofustis stercorihominis X Bacillus anthracis X Bacillus horti X Bacillus licheniformis X Bacillus nealsonii X Bacillus pumilus X Bacillus sp. BT1B_CT2 X Bacillus thuringiensis X Bacteroides galacturonicus X (phylogenetically in Clostridiales) Bacteroides pectinophilus X (phylogenetically in Clostridiales) Blautia wexlerae X X Brachyspira pilosicoli X Brevibacillus parabrevis X Clostridium aldenense X Clostridium beijerinckii X Clostridium carnis X Clostridium celatum X Clostridium favososporum X Clostridium hylemonae X Clostridium irregulare X Clostridium methylpentosum X Clostridium sp. D5 X X Clostridium sp. L2-50 X Clostridium sp. MT4 E X Clostridium sp. NML 04A032 X Clostridium sp. SS2/1 X Clostridium sp. YIT 12069 X Clostridium stercorarium X Clostridium xylanolyticum X Coprococcus sp. ART55/1 X Deferribacteres sp. X oral clone JV006 Desulfitobacterium frappieri X Eubacterium callanderi X Eubacterium siraeum X Exiguobacterium acetylicum X Gemmiger formicilis X Lachnospira multipara X Lachnospira pectinoschiza X Roseburia faecalis X Ruminococcus albus X

TABLE 6 Donor A, 45 species in 374 EtOH-resistant colonies sequenced OTU Anaerostipes_sp_3_2_56FAA Bacillus_anthracis Bacillus_cereus Bacillus_thuringiensis Blautia_producta Blautia_sp_M25 Clostridiales_sp_SSC_2 Clostridium_aldenense Clostridium_bartlettii Clostridium_bolteae Clostridium_celatum Clostridium_disporicum Clostridium_ghonii Clostridium_hathewayi Clostridium_lactatifermentans Clostridium_mayombei Clostridium_orbiscindens Clostridium_paraputrificum Clostridium_perfringens Clostridium_sordellii Clostridium_stercorarium Clostridium_straminisolvens Clostridium_tertium Coprobacillus_sp_D7 Coprococcus_catus Deferribacteres_sp_oral_clone_JV006 Dorea_formicigenerans Eubacterium_rectale Eubacterium_siraeum Eubacterium_sp_WAL_14571 Eubacterium_ventriosum Flexistipes_sinusarabici Fulvimonas_sp_NML_060897 Lachnospiraceae_bacterium_2_1_58FAA Lachnospiraceae_bacterium_3_1_57FAA Lachnospiraceae_bacterium_A4 Lachnospiraceae_bacterium_oral_taxon_F15 Moorella_thermoacetica Roseburia_faecalis Roseburia_hominis Ruminococcus_albus Ruminococcus_bromii Ruminococcus_gnavus Ruminococcus_sp_5_1_39BFAA Ruminococcus_torques

TABLE 7 Donor B, 26 species in 195 EtOH-resistant colonies sequenced OTU Bacillus_horti Blautia_wexlerae Chlamydiales_bacterium_NS11 Clostridiales_sp_SSC_2 Clostridium_bartlettii Clostridium_celatum Clostridium_disporicum Clostridium_ghonii Clostridium_oroticum Clostridium_paraputrificum Clostridium_perfringens Clostridium_sordellii Clostridium_sp_L2_50 Clostridium_sp_MT4_E Clostridium_straminisolvens Coprococcus_sp_ART55_1 Eubacterium_callanderi Eubacterium_rectale Eubacterium_ruminantium Gemmiger_formicilis Lachnospira_pectinoschiza Ruminococcus_albus Ruminococcus_gnavus Ruminococcus_obeum Ruminococcus_sp_5_1_39BFAA Ruminococcus_sp_K_1

TABLE 8 Donor C, 39 species in 416 EtOH-resistant colonies sequenced OTU Bacteroides_galacturonicus Bacteroides_pectinophilus Blautia_producta Blautia_sp_M25 Blautia_wexlerae Clostridiales_sp_SS3_4 Clostridiales_sp_SSC_2 Clostridium_bartlettii Clostridium_citroniae Clostridium_disporicum Clostridium_indolis Clostridium_orbiscindens Clostridium_paraputrificum Clostridium_sordellii Clostridium_sp_NML_04A032 Clostridium_sp_SS2_1 Clostridium_straminisolvens Clostridium_viride Clostridium_xylanolyticum Coprobacillus_sp_D7 Dorea_longicatena Eubacterium_rectale Eubacterium_ventriosum Hydrogenoanaerobacterium_saccharovorans Lachnospira_multipara Lachnospira_pectinoschiza Lachnospiraceae_bacterium_A4 Oscillibacter_sp_G2 Pseudoflavonifractor_capillosus Roseburia_hominis Roseburia_intestinalis Ruminococcus_albus Ruminococcus_lactaris Ruminococcus_obeum Ruminococcus_sp_5_1_39BFAA Ruminococcus_sp_K_1 Ruminococcus_torques Syntrophococcus_sucromutans

TABLE 9 Donor D, 12 species in 118 EtOH-resistant colonies sequenced OTU Blautia_luti Blautia_wexlerae Brachyspira_pilosicoli Clostridium_paraputrificum Collinsella_aerofaciens Coprobacillus_sp_D7 Desulfitobacterium_frappieri Eubacterium_rectale Moorella_thermoacetica Ruminococcus_gnavus Ruminococcus_obeum Ruminococcus_sp_K_1

TABLE 10 Donor E, 11 species in 118 EtOH-resistant colonies sequenced OTU Blautia_luti Blautia_wexlerae Brachyspira_pilosicoli Clostridium_paraputrificum Coprobacillus_sp_D7 Desulfitobacterium_frappieri Eubacterium_rectale Moorella_thermoacetica Ruminococcus_gnavus Ruminococcus_obeum Ruminococcus_sp_K_1

TABLE 11 Donor F, 54 OTUs in 768 EtOH-resistant colonies sequenced OTU Anaerofustis_stercorihominis Anaerostipes_sp_3_2_56FAA Bacillus_nealsonii Bacillus_sp_BT1B_CT2 Blautia_producta Butyrivibrio_crossotus Clostridiales_bacterium_SY8519 Clostridiales_sp_1_7_47 Clostridium_aldenense Clostridium_bartlettii Clostridium_bolteae Clostridium_butyricum Clostridium_citroniae Clostridium_clostridioforme Clostridium_disporicum Clostridium_favososporum Clostridium_glycolicum Clostridium_hathewayi Clostridium_indolis Clostridium_leptum Clostridium_mayombei Clostridium_nexile Clostridium_orbiscindens Clostridium_sordellii Clostridium_sp_7_2_43FAA Clostridium_sp_D5 Clostridium_sp_M62_1 Clostridium_sp_NML_04A032 Clostridium_spiroforme Clostridium_symbiosum Clostridium_tertium Coprobacillus_sp_29_1 Coprobacillus_sp_D7 Eubacterium_contortum Eubacterium_desmolans Eubacterium_ramulus Exiguobacterium_acetylicum Faecalibacterium_prausnitzii Lachnospiraceae_bacterium_2_1_58FAA Lachnospiraceae_bacterium_3_1_57FAA Lachnospiraceae_bacterium_5_1_57FAA Lachnospiraceae_bacterium_6_1_63FAA Lachnospiraceae_bacterium_oral_taxon_F15 Marvinbryantia_formatexigens Mycoplasma_amphoriforme Oscillibacter_sp_G2 Pseudoflavonifractor_capillosus Ruminococcus_gnavus Ruminococcus_hansenii Ruminococcus_obeum Ruminococcus_sp_5_1_39BFAA Ruminococcus_sp_ID8 Turicibacter_sanguinis

TABLE 12 Organisms grown from ethanol treated spore population on various media (See Example 5 for full media names and references). total number unique % unique Media reads OTUs OTUs M2GSC 93 33 0.35 M-BHI 66 26 0.39 Sweet B 74 23 0.31 GAM fructose 44 18 0.41 M2 mannitol 39 17 0.44 M2 soluble starch 62 16 0.26 M2 lactate 43 14 0.33 GAM FOS/Inulin 52 14 0.27 EYA 29 13 0.45 Mucin 19 12 0.63 M2 lactose 32 12 0.38 BHIS az1/ge2 35 12 0.34 BHIS CInM az1/ge2 24 11 0.46 GAM mannitol 41 11 0.27 BBA 29 10 0.34 Sulfite-polymyxin milk 48 9 0.19 Noack-Blaut Eubacterium agar 12 4 0.33 742 total analyzed

TABLE 13 Species identified as germinable and sporulatable by colony picking GAM + Sweet B + Sweet OTU BBA FOS/inulin M2GSC FOS/Inulin GAM Total Blautia producta 1 1 Clostridium bartlettii 4 1 5 Clostridium bolteae 2 5 1 8 Clostridium botulinum 5 5 Clostridium butyricum 37 43 8 1 33 122 Clostridium celatum 4 1 5 Clostridium clostridioforme 1 1 2 Clostridium disporicum 26 26 22 33 50 157 Clostridium glycolicum 4 9 14 27 Clostridium mayombei 2 2 4 Clostridium paraputrificum 8 8 33 16 6 71 Clostridium sordellii 14 14 Clostridium sp. 7_2_43FAA 1 1 Clostridium symbiosum 3 3 Clostridium tertium 1 1 2 (blank) 2 31 33 Totals 92 92 92 92 92 460

TABLE 15 Results of the prophylaxis mouse model and dosing information for the germinable, and sporulatable fractions. Average Average Weight on Day Clinical # Deaths 3 Relative Score Test Article Dose by Day 6 to Day −1 on Day 3 Vehicle NA 10 0.72 NA Naive NA 0 1.03 0 Donor B 0.2 mL of 10% 1 0.91 0.11 fecal suspension suspension Donor A 8.99*10{circumflex over ( )}7 Spore 0 1.02 0 Spore Prep Equivalents/dose germinable Donor A 7.46*10{circumflex over ( )}7 Spore 0 0.99 0 Spore Prep Equivalents/dose Sporulatable

Clinical score is based on a combined phenotypic assessment of the mouse's health on a scale of 0-4 in several areas including appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), and clinical signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).

TABLE 16 Bacterial OTUs associated with engraftment and ecological augmentation and establishment of a more diverse microbial ecology in patients treated with an ethanol treated spore preparation. Dominant Phylo- Spore OTU in genetic Forming Augmented OTU Clade OTU Ecology Bacteroides sp. 2_1_22 clade38 N Y Streptococcus anginosus clade60 N Prevotella intermedia clade81 N Prevotella nigrescens clade81 N Oribacterium sp. ACB7 clade90 N Prevotella salivae clade104 N Bacteroides intestinalis clade171 N Y Bifidobacterium dentium clade172 N Alcaligenes faecalis clade183 N Rothia dentocariosa clade194 N Peptoniphilus lacrimalis clade291 N Anaerococcus sp. gpac155 clade294 N Sutterella stercoricanis clade302 N Y Bacteroides sp. 3_1_19 clade335 N Y Parabacteroides goldsteinii clade335 N Bacteroides dorei clade378 N Y Bacteroides massiliensis clade378 N Lactobacillus iners clade398 N Granulicatella adiacens clade460 N Eggerthella sp. 1_3_56FAA clade477 N Gordonibacter pamelaeae clade477 N Finegoldia magna clade509 N Actinomyces nasicola clade523 N Streptobacillus moniliformis clade532 N Oscillospira guilliermondii clade540 N Orientia tsutsugamushi clade541 N Christensenella minuta clade558 N Clostridium oroticum clade96 Y Clostridium sp. D5 clade96 Y Clostridium glycyrrhizinilyticum clade147 Y Coprococcus comes clade147 Y Ruminococcus lactaris clade147 Y Ruminococcus torques clade147 Y Y Clostridiales sp. SS3/4 clade246 Y Clostridium hylemonae clade260 Y Clostridium aerotolerans clade269 Y Clostridium asparagiforme clade300 Y Y Clostridium sp. M62/1 clade300 Y Clostridium symbiosum clade300 Y Lachnospiraceae genomosp. C1 clade300 Y Blautia sp. M25 clade304 Y Y Blautia stercoris clade304 Y Ruminococcus hansenii clade304 Y Ruminococcus obeum clade304 Y Ruminococcus sp. 5_1_39BFAA clade304 Y Bryantella formatexigens clade309 Y Eubacterium cellulosolvens clade309 Y Clostridium sp. HGF2 clade351 Y Clostridium bartlettii clade354 Y Clostridium bifermentans clade354 Y Clostridium glycolicum clade354 Y Eubacterium tenue clade354 Y Dorea formicigenerans clade360 Y Dorea longicatena clade360 Y Lachnospiraceae bacterium clade360 Y 2_1_46FAA Lachnospiraceae bacterium clade360 Y Y 9_1_43BFAA Ruminococcus gnavus clade360 Y Clostridium hathewayi clade362 Y Blautia hydrogenotrophica clade368 Y Clostridiaceae bacterium END-2 clade368 Y Roseburia faecis clade369 Y Roseburia hominis clade370 Y Roseburia intestinalis clade370 Y Eubacterium sp. WAL 14571 clade384 Y Erysipelotrichaceae bacterium clade385 Y 5_2_54FAA Eubacterium biforme clade385 Y Eubacterium dolichum clade385 Y Coprococcus catus clade393 Y Acetivibrio ethanolgignens clade396 Y Anaerosporobacter mobilis clade396 Y Bacteroides pectinophilus clade396 Y Eubacterium hallii clade396 Y Eubacterium xylanophilum clade396 Y Anaerostipes caccae clade408 Y Clostridiales bacterium clade408 Y 1_7_47FAA Clostridium aldenense clade408 Y Clostridium citroniae clade408 Y Eubacterium hadrum clade408 Y Y Acetanaerobacterium elongatum clade439 Y Faecalibacterium prausnitzii clade478 Y Gemmiger formicilis clade478 Y Y Eubacterium ramulus clade482 Y Lachnospiraceae bacterium clade483 Y 3_1_57FAA_CT1 Lachnospiraceae bacterium A4 clade483 Y Y Lachnospiraceae bacterium DJF clade483 Y VP30 Holdemania filiformis clade485 Y Clostridium orbiscindens clade494 Y Pseudoflavonifractor capillosus clade494 Y Ruminococcaceae bacterium D16 clade494 Y Acetivibrio cellulolyticus clade495 Y Eubacterium limosum clade512 Y Anaerotruncus colihominis clade516 Y Clostridium methylpentosum clade516 Y Clostridium sp. YIT 12070 clade516 Y Hydrogenoanaerobacterium clade516 Y saccharovorans Eubacterium ventriosum clade519 Y Eubacterium eligens clade522 Y Lachnospira pectinoschiza clade522 Y Lactobacillus rogosae clade522 Y Y Clostridium leptum clade537 Y Eubacterium coprostanoligenes clade537 Y Ruminococcus bromii clade537 Y Clostridium viride clade540 Y Butyrivibrio crossotus clade543 Y Coprococcus eutactus clade543 Y Eubacterium ruminantium clade543 Y Eubacterium rectale clade568 Y Y Roseburia inulinivorans clade568 Y Butyricicoccus pullicaecorum clade572 Y Eubacterium desmolans clade572 Y Papillibacter cinnamivorans clade572 Y Sporobacter termitidis clade572 Y Clostridium lactatifermentans clade576 Y

TABLE 18 Reduction in the opportunistic pathogen or pathobiont load by ethanol treated spores. Pre- treatment Day 5 Day 14 Day 25 Klebsiella (% of total reads) 20.27% 1.32% 7.62% 0.00% Fusobacterium (% total of reads) 19.14% 3.01% 0.01% 0.00%

TABLE 19 Changes in Enterobacteria as a function of treatment measured on Simmons Citrate Agar Pretreatment titer Day 25 titer Patient Organism (cfu/g) (cfu/g) 1 Klebsiella pneumoniae 9 × 10⁶ 1 × 10³ 1 Klebsiella sp. Co9935 4 × 10⁶ 1 × 10³ 1 Escherichia coli 7 × 10⁶ 1 × 10⁶ 2 Klebsiella sp. Co9935 4 × 10⁶ 1 × 10³ 4 Klebsiella pneumoniae 3 × 10⁸ <1 × 10⁴  4 Klebsiella sp. Co9935 6 × 10⁷ <1 × 10⁴  5 Klebsiella pneumoniae 1 × 10⁶ <1 × 10⁴ 

TABLE 20 Augmentation of Bacteroides as a function of bacterial composition treatment of Patient 1 Pretreatment titer Day 25 titer Media Bacteroides species (cfu/g) (cfu/g) BBE B. fragilis group <2 × 10⁴ 3 × 10⁸  PFA All Bacteroides <2 × 10⁷ 2 × 10¹⁰

TABLE 21 Bacteroides spp. post-treatment with the ethanol treated spore preparation based full-length 16S rDNA sequences of isolated strains % of total Bacteroides cfu Species (1.58E10 cfu/g) Bacteroides sp. 4_1_36  63% Bacteroides cellulosilyticus  14% Bacteroides sp. 1_1_30  14% Bacteroides uniformis  4.8% Bacteroides ovatus  1.7% Bacteroides dorei 0.91% Bacteroides xylanisolvens 0.83% Bacteroides sp. 3_1_19 0.23%

TABLE 22 Titers (in cfu/g) of imipenem-resistant M. morganii, P. rettgeri and P. pennerii from Patients B, D & E Patient Organism Pretreatment titer Day 28 titer * Patient 2 M. morganii 1 × 10⁴  6 × 10² Patient 2 P. rettgeri 9 × 10³ <5 × 10¹ Patient 4 M. morganii 2 × 10⁴ <5 × 10¹ Patient 4 P. pennerii 2 × 10⁴ <5 × 10¹ Patient 5 M. morganii 5 × 10³ <5 × 10¹ * Limit of detection based on plating 200 uL of 10% wt/vol suspension is 5 × 10¹

TABLE YYY Species identified as germinable by 16S colony pick approach Clostridium_paraputrificum Clostridium_disporicum Clostridium_glycolicum Clostridium_bartlettii Clostridium_butyricum Ruminococcus_bromii Lachnospiraceae_bacterium_2_1_58FAA Eubacterium_hadrum Turicibacter_sanguinis Lachnospiraceae_bacterium_oral_taxon_F15 Clostridium_perfringens Clostridium_bifermentans Roseburia_sp_11SE37 Clostridium_quinii Ruminococcus_lactaris Clostridium_botulinum Clostridium_tyrobutyricum Blautia_hansenii Clostridium_kluyveri Clostridium_sp_JC122 Clostridium_hylemonae Clostridium_celatum Clostridium_straminisolvens Clostridium_orbiscindens Roseburia_cecicola Eubacterium_tenue Clostridium_sp_7_2_43FAA Lachnospiraceae_bacterium_4_1_37FAA Eubacterium_rectale Clostridium_viride Ruminococcus_sp_K_1 Clostridium_symbiosum Ruminococcus_torques Clostridium_algidicarnis

TABLE ZZZ Species identified as sporulatable by 16S NGS approach Clostridium_paraputrificum Clostridium_bartlettii Lachnospiraceae_bacterium_2_1_58FAA Clostridium_disporicum Ruminococcus_bromii Eubacterium_hadrum Clostridium_butyricum Roseburia_sp_11SE37 Clostridium_perfringens Clostridium_glycolicum Clostridium_hylemonae Clostridium_orbiscindens Ruminococcus_lactaris Clostridium_symbiosum Lachnospiraceae_bacterium_oral_taxon_F15 Blautia_hansenii Turicibacter_sanguinis Clostridium_straminisolvens Clostridium_botulinum Lachnospiraceae_bacterium_4_1_37FAA Roseburia_cecicola Ruminococcus_sp_K_1 Clostridium_bifermentans Eubacterium_rectale Clostridium_quinii Clostridium_viride Clostridium_kluyveri Clostridium_tyrobutyricum Oscillibacter_sp_G2 Clostridium_sp_JC122 Lachnospiraceae_bacterium_3_1_57FAA Clostridium_aldenense Ruminococcus_torques Clostridium_sp_7_2_43FAA Clostridium_celatum Eubacterium_sp_WAL_14571 Eubacterium_tenue Lachnospiraceae_bacterium_5_1_57FAA Clostridium_clostridioforme Clostridium_sp_YIT_12070 Blautia_sp_M25 Anaerostipes_caccae Roseburia_inulinivorans Clostridium_sp_D5 Clostridium_asparagiforme Coprobacillus_sp_D7 Clostridium_sp_HGF2 Clostridium_citroniae Clostridium_difficile Oscillibacter_valericigenes Clostridium_algidicarnis 

1-99. (canceled)
 100. A therapeutic composition formulated for oral administration comprising a purified population of bacteria, an enteric coating, and a capsule, wherein the purified population of bacteria consist of spore-forming bacteria in the form of spores.
 101. The therapeutic composition of claim 100, wherein the spore-forming bacteria consists essentially of germinable bacterial spores, sporulatable bacterial spores, or both.
 102. The therapeutic composition of claim 100, wherein the composition is derived from a fecal material subjected to ethanol treatment or heat treatment.
 103. The therapeutic composition of claim 100, wherein the composition augments a titer of one or more non-pathogenic Bacteroides selected from the group consisting of Bacteroides sp. 4_1_36, Bacteroides cellulosilyticus, Bacteroides sp. 1_1_30, Bacteroides uniformis, Bacteroides ovatus, Bacteroides dorei, Bacteroides xylanisolvens, and Bacteroides sp. 3_1_19; and wherein the non-pathogenic Bacteroides titer is augmented in the gastrointestinal tract of the subject.
 104. The therapeutic composition of claim 102, wherein the fecal material is a 10 to 20% fecal suspension.
 105. The therapeutic composition of claim 102, wherein the fecal material is obtained from a validated mammalian donor subject not having a detectable level of a pathogen or a pathobiont prior to production of the fecal material.
 106. The therapeutic composition of claim 102, wherein the fecal material is obtained from a validated mammalian donor subject not having a detectable level of a blood-borne pathogen or a fecal bacterial pathogen prior to production of the fecal material.
 107. The therapeutic composition of claim 100, wherein the composition comprises at least 1×10⁴ colony forming units of the spore-forming bacteria per dose of the composition.
 108. The therapeutic composition of claim 100, wherein the capsule is a delayed release capsule.
 109. A method of treating or reducing recurrence of Clostridium difficile infection (CDI) or a symptom thereof in a subject in need thereof, comprising administering the therapeutic composition of claim 100 to the subject in an amount effective to treat or reduce recurrence of the CDI or a symptom thereof.
 110. The method of claim 109, wherein at least one type of spore-forming bacteria in the composition is not detectably present in the gastrointestinal tract of the subject prior to administration of the composition to the subject.
 111. The method of claim 109, wherein administering the composition results in augmenting growth of at least one type of bacteria not detectably present in the composition or in the gastrointestinal tract prior to administration.
 112. The method of claim 109, wherein administering the composition results in at least two of: i) reduction or elimination of at least one pathogen and/or pathobiont present in the gastrointestinal tract of the subject; ii) engraftment in the gastrointestinal tract of the subject of at least one type of spore-forming bacteria present in the composition; and iii) augmentation of at least one type of spore-forming or non-spore forming bacteria in the gastrointestinal tract of the subject that is not present in the composition.
 113. The method of claim 109, wherein the subject has received one or more doses of an antibiotic therapy.
 114. A method of producing the therapeutic composition of claim 100, comprising: a) providing a fecal material; and b) subjecting the fecal material to a treatment step resulting in purification of a purified population of bacteria consisting of spore-forming bacteria in the form of spores.
 115. The method of claim 114, further comprising c) isolating the purified population of bacteria consisting of spore-forming bacteria.
 116. The method of claim 114, wherein the fecal material is feces or materials isolated from the various segments of the small and large intestines.
 117. The method of claim 114, wherein the treatment step comprises one or more solvent treatments.
 118. The method of claim 114, wherein the treatment step comprises one or more chromatographic treatments.
 119. The method of claim 114, wherein the treatment step comprises one or more mechanical treatments. 